Recovery of recombinant human parainfluenza virus type 2 (HPIV2) from CDNA and use of recombinant HPIV2 in immunogenic compositions and as vectors to elicit immune responses against PIV and other human pathogens

ABSTRACT

Recombinant human parainfluenza virus type 2 (HPIV2) viruses and related immunogenic compositions and methods are provided. The recombinant HPIV2 viruses, including HPIV2 chimeric and chimeric vector viruses, provided according to the invention are infectious and attenuated in permissive mammalian subjects, including humans, and are useful in immunogenic compositions for eliciting an immune responses against one or more PIVs, against one or more non-PIV pathogens, or against a PIV and a non-PIV pathogen. Also provided are isolated polynucleotide molecules and vectors incorporating a recombinant HPIV2 genome or antigenome.

CROSS-REFERENCE PARAGRAPH

The present application is a divisional of application Ser. No.10/667,141, filed on Sep. 18, 2003, now U.S. Pat No. 7,820,181 which isa regular utility application of Provisional Application No. 60/412,053,filed on Sep. 18, 2002, the entire contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Human parainfluenza viruses (HPIVs) are important pathogens in humanpopulations, causing severe lower respiratory tract infections ininfants and young children. HPIV1 and HPIV2 are the principal etiologicagents of laryngotracheobronchitis (croup), and can also cause pneumoniaand bronchiolitis (Chanock et al., Parainfluenza Viruses., p. 1341-1379,In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin,B. Roizman, and S. E. Straus (eds.) Fields Virology, 4th ed., Vol. 1,Lippincott Williams & Wilkins, Philadelphia, 2001). HPIV3 ranks secondafter respiratory syncytial virus (RSV) as a leading cause ofhospitalization for viral lower respiratory tract disease in infants andyoung children (Collins et al., 3rd ed. In “Fields Virology,” B. N.Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P.Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1205-1243.Lippincott-Raven Publishers, Philadelphia, 1996; Crowe et al., Vaccine13:415-421, 1995; Marx et al., J. Infect. Dis. 176:1423-1427, 1997).

HPIVs are also important causes of respiratory tract disease in adults.Collectively, HPIV1, HPIV2, and HPIV3 have been identified through a 20year study as responsible etiologic agents for approximately 18% ofhospitalizations for pediatric respiratory tract disease (Murphy et al.,Virus Res. 11: 1-15, 1988). HPIVs have also been implicated in asignificant proportion of cases of virally-induced middle ear effusionsin children with otitis media (Heikkinen et al., N. Engl. J. Med.340:260-4, 1999).

Despite considerable efforts to develop effective vaccines againstHPIVs, no vaccines have yet been approved for any HPIV serotype, nor forameliorating HPIV related illnesses. The most promising prospects todate are live attenuated vaccine viruses since these have been shown tobe efficacious in non-human primates even in the presence of passivelytransferred antibodies, an experimental situation that simulates thatpresent in the very young infant who possesses maternally acquiredantibodies (Crowe et al., Vaccine 13:847-855, 1995; Durbin et al., J.Infect. Dis. 179:1345-1351, 1999). Two live attenuated HPIV3 vaccinecandidates, a temperature-sensitive (ts) derivative of the wild typeHPIV3 JS strain (designated HP1V3cp45) and a bovine PIV3 (BPIV3) strain,are undergoing clinical evaluation (Karron et al., Pediatr. Infect. Dis.J. 15:650-654, 1996; Karron et al., J. Infect. Dis. 171:1107-1114,1995a; Karron et al., J. Infect. Dis. 172, 1445-1450, 1995b). The liveattenuated PIV3cp45 vaccine candidate was derived from the JS strain ofHPIV3 via serial passage in cell culture at low temperature and has beenfound to be protective against HPIV3 challenge in experimental animalsand to be satisfactorily attenuated, genetically stable, and immunogenicin seronegative human infants and children (Belshe et al, J. Med. Virol.10:235-242, 1982; Belshe et al., Infect. Immun. 37:160-5, 1982; Clementset al., J. Clin. Microbiol. 29:1175-82, 1991; Crookshanks et al., J.Med. Virol. 13:243-9, 1984; Hall et al., Virus Res. 22:173-184, 1992;Karron et al., J. Infect. Dis. 172:1445-1450, 1995b). Because theseHPIV3 candidate vaccine viruses are biologically derived, there are noproven methods for adjusting the level of attenuation should this befound necessary from ongoing clinical trials.

To facilitate development of HPIV vaccines, recombinant DNA technologyhas recently made it possible to recover infectious negative-strandedRNA viruses from cDNA (for a review, see Conzelmann, J. Gen. Virol.77:381-89, 1996). In this context, recombinant rescue of infectiousvirus has been reported for respiratory syncytial virus (RSV), rabiesvirus (RaV), canine distemper virus, mumps virus, infectioushematopoietic necrosis virus, simian virus 5 (SV5), rinderpest virus,Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), measlesvirus (MeV), and Sendai virus (murine parainfluenza virus type 1(MPIV1)) from cDNA-encoded genomic or antigenomic RNA in the presence ofessential viral proteins (see, e.g., Garcin et al., EMBO J.14:6087-6094, 1995; Lawson et al., Proc. Natl. Acad. Sci. U.S.A.92:4477-81, 1995; Radecke et al., EMBO J. 14:5773-5784, 1995; Schnell etal., EMBO J. 13:4195-203, 1994; Whelan et al., Proc. Natl. Acad. Sci.U.S.A. 92:8388-92, 1995; Hoffman et al., J. Virol. 71:4272-4277, 1997;Kato et al., Genes to Cells 1:569-579, 1996, Roberts et al., Virology247:1-6, 1998; Baron et al., J. Virol. 71:1265-1271, 1997; InternationalPublication No. WO 97/06270; Collins et al., Proc. Natl. Acad. Sci.U.S.A. 92:11563-11567, 1995; Clarke et al., J. Virol. 74:4831-4838,2000; Biacchesi et al., J. Virol. 74:11247-11253, 2000; Gassen et al.,J. Virol. 74:10737-10744, 2000; U.S. patent application Ser. No.08/892,403, filed Jul. 15, 1997 (corresponding to publishedInternational Application No. WO 98/02530 and priority U.S. ProvisionalApplication Nos. 60/047,634, filed May 23, 1997, 60/046,141, filed May9, 1997, and 60/021,773, filed Jul. 15, 1996); U.S. patent applicationSer. No. 09/291,894, filed on Apr. 13, 1999; International ApplicationNo. PCT/US00/09695, filed Apr. 12, 2000 (which claims priority to U.S.Provisional Patent Application Ser. No. 60/129,006, filed Apr. 13,1999); International Application No. PCT/US00/17755, filed Jun. 23, 2000(which claims priority to U.S. Provisional Patent Application Ser. No.60/143,132, filed by Buchholz et al. on Jul. 9, 1999); Juhasz et al., J.Virol. 71:5814-5819, 1997; He et al. Virology 237:249-260, 1997; Peterset al. J. Virol. 73:5001-5009, 1999; Baron et al. J. Virol.71:1265-1271, 1997; Whitehead et al., Virology 247:232-9, 1998a;Whitehead et al., J. Virol. 72:4467-4471, 1998b; Jin et al. Virology251:206-214, 1998; Buchholz et al. J. Virol. 73:251-259, 1999; andWhitehead et al., J. Virol. 73:3438-3442, 1999, each incorporated hereinby reference in its entirety for all purposes).

Additional publications in the field of the invention report recovery ofrecombinant parainfluenza viruses (PIVs), specifically HPIV1, HPIV2,HPIV3, and BPIV3 (see, e.g., Durbin et al., Virology 235:323-332, 1997;Schmidt et al., J. Virol. 74:8922-8929, 2000; Kawano et al., Virology284:99-112, 2001; U.S. patent application Ser. No. 09/083,793, filed May22, 1998 (corresponding to U.S. Provisional Application No. 60/059,385,filed Sep. 19, 1997); U.S. Provisional Application No. 60/331,961, filedNov. 21, 2001; and U.S. Provisional Application No. 60/047,575, filedMay 23, 1997 (corresponding to International Publication No. WO98/53078), each incorporated herein by reference). Some of these reportsfurther address genetic manipulation of viral cDNA clones to determinethe genetic basis of phenotypic changes in biological mutants, forexample, which mutations in a biological mutant HPIV3 JS cp45 virusspecify its ts, ca and att phenotypes, and which gene(s) or genomesegment(s) of BPIV specify its attenuation phenotype. Additionally,certain of these reports and related publications discuss constructionof novel PW vaccine candidates having a wide range of differentmutations, as well as methods for evaluating the level of attenuation,immunogenicity and phenotypic stability exhibited by such recombinantvaccine candidates (see also, U.S. application Ser. No. 09/586,479,filed Jun. 1, 2000 (corresponding to U.S. Provisional Patent ApplicationSer. No. 60/143,134, filed on Jul. 9, 1999); and U.S. patent applicationSer. No. 09/350,821, filed by Durbin et al. on Jul. 9, 1999, eachincorporated herein by reference).

Thus, infectious wild type recombinant PIVs, (r)PIVs, as well as anumber of ts and otherwise modified derivatives, have now been recoveredfrom cDNA. Reverse genetics systems have been used to generateinfectious virus bearing defined mutations that specify attenuation andother desirable phenotypes, and to study the genetic basis ofattenuation and other phenotypic changes in existing vaccine candidateviruses. For example, in HPIV3, the three amino acid substitutions foundin the L gene of cp45, singularly or in combination, have been found tospecify the ts and attenuation phenotypes. Additional ts and otherattenuating mutations can be introduced in other regions of the HPIV3genome.

In addition, a chimeric PIV1 vaccine candidate has been generated usingthe PIV3 cDNA rescue system by replacing the PIV3 RN and F open readingframes (ORFs) with those of PIV1 in a PIV3 full-length cDNA, thatoptionally contains selected attenuating mutations. Exemplaryrecombinant chimeric viruses derived from these cDNA-based methodsinclude a HPIV3-1 recombinant bearing all three identified mutations inthe L gene, rP1V3-1.cp45L (Skiadopoulos et al., J. Virol. 72:1762-8,1998; Tao et al., J. Virol. 72:2955-2961, 1998; Tao et al., Vaccine17:1100-1108, 1999, incorporated herein by reference). rHPIV3-1.cp45Lwas attenuated in hamsters and induced a high level of resistance tochallenge with HPIV1. Yet another recombinant chimeric virus, designatedrHPIV3-1 cp45, has been produced that contains 12 of the 15 cp45mutations, i.e, excluding the mutations that occur in HN and F. Thisrecombinant vaccine candidate is highly attenuated in the upper andlower respiratory tract of hamsters and non-human primates and induces ahigh level of protection against HPIV1 infection (Skiadopoulos et al.,Vaccine 18:503-510, 1999; Skiadopoulos et al., Virology 297:136-152,2002, each incorporated herein by reference). However, for use againstHPIV1, the infection and attendant immunogenicity of chimeric HPIV3-1vaccine candidates against HPIV1 challenge is dampened in hosts thatexhibit immune recognition of HPIV3.

Recently, a number of studies have focused on the possible use of viralvectors to express foreign antigens toward the goal of developingvaccines against a pathogen for which other vaccine alternatives havenot yet proven successful. In this context, a number of reports suggestthat foreign genes may be successfully inserted into a recombinantnegative strand RNA virus genome or antigenome with varying effects(Bukreyev et al., J. Virol. 70:6634-41, 1996; Bukreyev et al., Proc.Natl. Acad. Sci. U.S.A. 96:2367-72, 1999; Finke et al. J. Virol.71:7281-8, 1997; Hasan et al., J. Gen. Virol. 78:2813-20, 1997; He etal., Virology 237:24960, 1997; Jin et al., Virology 251:206-14, 1998;Johnson et al., J. Virol. 71:5060-8, 1997; Kahn et al., Virology254:81-91, 1999; Kretzschmar et al., J. Virol. 71:5982-9, 1997;Mebatsion et al., Proc. Natl. Acad. Sci. U.S.A. 93:7310-4, 1996; Moriyaet al., FEBS Lett. 425:105-11, 1998; Roberts et al., J. Virol.73:3723-32, 1999; Roberts et al., J. Virol. 72:4704-11, 1998; Roberts etal., Virology 247:1-6, 1998; Sakai et al., FEBS Lett. 456:221-226, 1999;Schnell et al., Proc. Natl. Acad. Sci. U.S.A. 93:11359-65, 1996a;Schnell et al., J. Virol. 70:2318-23, 1996b; Schnell et al., Cell90:849-57, 1997; Singh et al., J. Gen. Virol. 80:101-6, 1999; Singh etal., J. Virol. 73:4823-8, 1999; Spielhofer et al., J. Virol. 72:2150-9,1998; Yu et al., Genes to Cells 2:457-66 et al., 1999; Duprex et al., J.Virol. 74:7972-7979, 2000; Subash et al., J. Virol. 74:9039-9047, 2000;Krishnamurthy et al., Virology 278:168-182, 2000; Rose et al., J. Virol.74:10903-10910, 2000; Tao et al., J. Virol. 74:6448-6458, 2000;McGettigan et al., J. Virol. 75:8724-8732, 2001; McGettigan et al., J.Virol. 75:4430-4434, 2001; Kahn et al., J. Virol. 75:11079-11087, 2001;Stope et al., J. Virol. 75:9367-9377, 2001; Huang et al., J. Gen. Virol.82:1729-1736, 2001; Skiadopoulos et al., J. Virol. 75:10498-10504, 2001;Bukreyev et al., J. Virol. 75:12128-12140, 2001; U.S. patent applicationSer. No. 09/614,285, filed Jul. 12, 2000 (corresponding to U.S.Provisional Patent Application Ser. No. 60/143,425, filed on Jul. 13,1999), each incorporated herein by reference). When inserted into theviral genome under the control of viral transcription gene-start andgene-end signals, the foreign gene may be transcribed as a separate mRNAand yield significant protein expression. Surprisingly, in most casesthe foreign sequence has been reported to be relatively stable andcapable of expressing functional protein during numerous passages invitro.

In order to successfully develop vectors for vaccine use, however, it isinsufficient to simply demonstrate a high, stable level of proteinexpression. For example, this has been possible since the early-to-mid1980s with recombinant vaccinia viruses and adenoviruses, and yet thesevectors have proven to be disappointing tools for developing vaccinesfor human use. Similarly, most nonsegmented negative strand viruses thathave been developed as vectors have not yet been shown to be amenablefor human vaccine use. Examples in this context include vesicularstomatitis virus, an ungulate pathogen with no history of administrationto humans except for a few laboratory accidents; Sendai virus, a mousepathogen with no history of administration to humans; simian virus 5, acanine pathogen with no history of intentional administration to humans;and an attenuated strain of measles virus which must be administeredsystemically and would be neutralized by measles-specific antibodiespresent in nearly all humans due to maternal antibodies and widespreaduse of a licensed measles vaccine. Furthermore, some of these priorvector candidates have adverse effects, such as immunosuppression, whichare directly inconsistent with their use as vectors. Thus, one mustidentify vectors whose growth characteristics, tropisms, and otherbiological properties make them appropriate as vectors for human use. Itis further necessary to develop a viable immunization strategy,including efficacious timing and route of administration.

Proposed mononegaviruses for use as vaccine vectors include measles,mumps, VSV, and rabies viruses. However, measles virus has limitationsrelating to its potential use as a vaccine vector. For example, measlesvirus has been considered for use a vector for the protective antigen ofhepatitis B virus (Singh et al., J. Virol. 73:4823-8, 1999). However,this combined measles virus-hepatitis B virus vaccine candidate couldonly be administered after nine months of age, on a schedule comparableto the indicated schedule for the licensed measles virus vaccine,whereas the current hepatitis B virus vaccine is recommended for use inearly infancy. This is because the currently licensed measles vaccine isadministered parenterally and is sensitive to neutralization andimmunosuppression by maternal antibodies, and therefore is not effectiveif administered before 9-15 months of age. Thus, measles virus is a poorvector for antigens of pathogenic agents that cause disease in earlyinfancy, such as RSV and the HPIVs.

The attenuated measles virus vaccine has been associated with alteredimmune responses and excess mortality when administered at increaseddosages, which may be due at least in part to virus-inducedimmunosuppression that is a common feature of natural measles virusinfection. This indicates that even an attenuated measles virus may notbe suitable for vaccine vector use. Furthermore, the use of measlesvirus as a vector would be inconsistent with the global effort toeradicate this pathogen. Indeed, for these reasons it would be desirableto end the use of live measles virus and replace the present measlesvirus vaccine with a suitable non-measles vector that expresses measlesvirus protective antigens.

Rabies virus, a rare cause of infection of humans, has been consideredfor use as a vector (Mebatsion et al., Proc. Natl. Acad. Sci. USA93:7310-4, 1996), but it is unlikely that a virus that is so highlyfatal as rabies for humans could be developed for use as a liveattenuated virus vector. Moreover, immunity to the rabies virus, whichis not a ubiquitous human pathogen, is not needed for the generalpopulation. In addition, in some circumstances it may be desirable forthe vector to be capable of eliciting a multispecific immune responseagainst both the vector virus and the pathogen for which the vector isused as a carrier of antigenic determinants. While measles virus is lesspathogenic than the rabies virus, infection by either of this vectorcandidate can yield undesirable results. Measles virus establishes aviremia with widespread infection and associated rash and theabove-mentioned immunosuppression. Mild encephalitis during measlesinfection is not uncommon. Measles virus is also associated with a rareprogressive fatal neurological disease called subacute sclerosingencephalitis.

In contrast to such vector candidates as rabies and measles, PIVinfection and disease is typically more limited, in part by confinementof infection to the respiratory tract. Viremia and spread to secondarysites can occur in severely immunocompromised subjects, but this is nota typical effect of PIV infection. Acute respiratory tract disease isthe only disease associated with PIVs. Thus, the use of PIVs as vectorswill, on the basis of their biological characteristics, avoidcomplications such as interaction of virus with peripheral lymphocytes,leading to immunosuppression, or infection of secondary organs such asthe testes or central nervous system, leading to other complications.These characteristics also render PIV a better vector candidate forsuccessful immunization, which can be achieved more easily andeffectively via alternate routes, such as direct administration to therespiratory tract, compared to immunization with vectors that requireparental administration.

Among a host of human pathogens for which a vector-based vaccineapproach may be desirable is the measles virus. A live attenuatedvaccine has been available for more than three decades and has beenlargely successful in eradicating measles disease in the United States.However, the World Health Organization estimates that more than 45million cases of measles still occur annually, particularly indeveloping countries, and the virus contributes to approximately onemillion deaths per year. One reason for the persistence of this diseaseis the inefficacy of current vaccine formulations to overcome maternalantibodies that inactivate the current vaccine.

Measles virus is a member of the Morbillivirus genus of theParamyxoviridae family (Griffin et al., In “Fields Virology”, B. N.Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P.Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312.Lippincott-Raven Publishers, Philadelphia, 1996). It is one of the mostcontagious infectious agents known to man and is transmitted from personto person via the respiratory route (Griffin et al., In “FieldsVirology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L.Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp.1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The measlesvirus has a complex pathogenesis, involving replication in both therespiratory tract and various systemic sites (Griffin et al., In “FieldsVirology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L.Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp.1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). Measlesvirus is discussed here as an exemplary pathogen for which a liveattenuated vector vaccine is particularly desired. For reasons discussedin further detail herein below, a measles vaccine based on a recombinantHPIV2 vector system would satisfy a long-felt need in the art andfulfill an urgent need for additional effective vector systems togenerate vaccines against other pathogens as well.

Although both mucosal IgA and serum IgG measles virus-specificantibodies can participate in the control of measles virus, the absenceof measles virus disease in very young infants possessingmaternally-acquired measles virus-specific antibodies identifies serumantibodies as a major mediator of resistance to disease (Griffin et al.,In “Fields Virology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M.Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus,Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia,1996). The two measles virus glycoproteins, the hemagglutinin (HA) andfusion (F) proteins, are the major neutralization and protectiveantigens (Griffin et al., In “Fields Virology”, B. N. Fields, D. M.Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B.Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-RavenPublishers, Philadelphia, 1996).

The currently available live attenuated measles vaccine is administeredby a parenteral route (Griffin et al., In “Fields Virology”, B. N.Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P.Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312.Lippincott-Raven Publishers, Philadelphia, 1996). Both the wild typemeasles virus and the vaccine virus are very readily neutralized byantibodies, and the measles virus vaccine is rendered non-infectious byeven very low levels of maternally-acquired measles virus-specificneutralizing antibodies (Halsey et al., N. Engl. J. Med. 313:544-9,1985; Osterhaus et al., Vaccine 16:1479-81, 1998). Thus, the vaccinevirus is not given until the passively-acquired maternal antibodies havedecreased to undetectable levels. In the United States, measles virusvaccine is not given until 12 to 15 months of age, a time when almostall children are readily infected with the measles virus vaccine.

Recent developments in the field of negative stranded RNA viral vaccineshave involved the use of HPIV3-based vaccine vectors to deliverantigenic determinants of heterologous pathogens, including heterologousHPIVs. In particular, recombinant HPIV3 vaccine candidates have beendisclosed that use a HPIV3 “vector” genome or antigenome combined withone or more heterologous genes of a different HPIV, or of a non-PIVpathogen to form a chimeric, bivalent or multivalent, HPIV3-basedvaccine candidate (see, e.g., Durbin et al., Virology 235:323-332, 1997;Skiadopoulos et al., J. Virol. 72:1762-1768, 1998; Skiadopoulos et al.,J. Virol. 73:1374-1381, 1999; Tao et al., Vaccine 19:3620-3631, 2001;Durbin et al., J. Virol. 74:6821-6831, 2000; U.S. patent applicationSer. No. 09/083,793, filed May 22, 1998; U.S. patent application Ser.No. 09/458,813, filed Dec. 10, 1999; U.S. patent application Ser. No.09/459,062, filed Dec. 10, 1999; U.S. Provisional Application No.60/047,575, filed May 23, 1997 (corresponding to InternationalPublication No. WO 98/53078); U.S. Provisional Application No.60/059,385, filed Sep. 19, 1997; U.S. Provisional Application No.60/170,195 filed Dec. 10, 1999; U.S. patent application Ser. No.09/733,692, filed Dec. 8, 2000 (corresponding to InternationalPublication No. WO 01/42445A2), each incorporated herein by reference.The recombinant chimeric HPIV3 viruses are engineered to incorporate oneor more heterologous donor sequences, typically supernumerary sequences,encoding one or more antigenic determinants of a different PIV orheterologous pathogen to produce an infectious, chimeric, bivalent ormultivalent virus or subviral particle. In this manner, candidateHPIV3-based chimeric vaccine viruses can be made to elicit an immuneresponse against one or more PIVs or a polyspecific response against aselected PIV and a non-PIV pathogen in a mammalian host susceptible toinfection therefrom. Various modifications to chimeric HPIV3 vaccinecandidates were reported to yield desired phenotypic effects, such asattenuation. Comparable disclosure has been provided for recombinant andchimeric recombinant HPIV1 vaccine candidates (see, U.S. ProvisionalApplication No. 60/331,961, filed Nov. 21, 2001, incorporated herein byreference).

Although there have been numerous advances toward development ofeffective immunogenic compositions against HPIVs and other pathogens,including RSV and measles virus, there remains a clear need in the artfor additional tools and methods to engineer safe and effectiveimmunogenic compositions to alleviate the serious health problemsattributable to these pathogens, particularly among young infants. Amongthe remaining challenges in this context is the need for additionaltools to generate suitably attenuated, immunogenic and geneticallycandidates for use in diverse clinical settings against one or morepathogens. Additional challenges arise from the fact that HPIV1, HPIV2,and HPIV3 represent distinct viral serotypes, that do not elicitsignificant cross-immunity. Accordingly, there is an urgent need in theart for an effective immunogenic compositions to immunize againstmultiple HPIV serotypes. To facilitate these goals, existing methods foridentifying and incorporating attenuating mutations into recombinantstrains and for developing vector-based immunogenic compositions andmethods must be expanded. In this context, it is particularly desirableto develop a method for recovery and genetic manipulation of HPIV2, togenerate immunogenic compositions against this important human PIV, andto provide additional tools to generate novel vectors and immunizationmethods. Surprisingly, the present invention satisfies these needs andfulfills additional objects and advantages as described herein below.

SUMMARY OF THE INVENTION

The instant invention provides methods and compositions for recoveringinfectious, recombinant human parainfluenza virus type 2 (HPIV2). Theinvention also provides novel tools and methods for introducing defined,predetermined structural and phenotypic changes into an infectious HPIV2candidate for use in immunogenic compositions and methods.

In one embodiment of the invention, methods are provided for producingan infectious, self-replicating, recombinant human parainfluenza virustype 2 (HPIV2) from one or more isolated polynucleotide moleculesencoding the virus. The methods generally involve coexpressing in a cellor cell-free system one or more expression vector(s) comprising apolynucleotide molecule that encodes a partial or complete, recombinantHPIV2 genome or antigenome and one or more polynucleotide moleculesencoding PIV N, P and L proteins, so as to produce an infectious HPIV2particle.

Typically, the polynucleotide molecule that encodes the recombinantHPIV2 genome or antigenome is a cDNA. Thus, the invention is directed inmore detailed aspects to such novel polynucleotides and theirequivalents that encode a recombinant HPIV2, as disclosed herein.Likewise, the invention embraces expression vectors and constructs thatincorporate a polynucleotide molecule encoding a recombinant HPIV2genome or antigenome.

The HPIV2 genome or antigenome, and the N, P, and L proteins may all beproduced from a single expression vector. More typically, the genome orantigenome is produced by a separate expression vector, and the N, P,and L proteins are produced by one, two, or three additional expressionvector(s). In certain embodiments, one or more of the N, P and Lproteins is supplied by expression of a recombinant HPIV genome orantigenome of the invention, or by coinfection with the same ordifferent PIV. In alternate embodiments, one or more of the N, P and Lproteins are from a heterologous PIV (e.g., HPIV1 or HPIV3).

The invention further embraces infectious, recombinant, self-replicatingviral particles produced according to the foregoing methods, whichparticles include complete viruses as well as viruses that lack one ormore non-essential protein(s) or non-essential portion(s) (e.g., acytoplasmic, transmembrane or extracellular domain) of a viral protein.Viruses of the invention that lack one or more such non-essentialcomponent (e.g., a gene or genome segment from the PIV V open readingframe (ORF) or an intergenic or other non-coding or non-essential genomecomponent) are referred to herein as incomplete viruses or “subviralparticles.” Exemplary subviral particles may lack any selectedstructural element, e.g., a gene, gene segment, protein, proteinfunctional domain, etc., that is present in a complete virus (i.e, anassembled virion including a complete genome or antigenome, nucleocapsidand envelope). For example, a subviral particle of the invention maycomprise an infectious nucleocapsid containing a genome or antigenome,and the products of N, P, and L genes. Other subviral particles areproduced by partial or complete deletions or substitutions ofnon-essential genes and/or their products among other non-essentialstructural elements.

Complete viruses and subviral particles produced according to themethods of the invention are typically infectious and self-replicativethrough multiple rounds of replication in a mammalian host amenable toinfection by PIV, including various in vitro mammalian cell populations,in vivo animal models widely known and accepted in the art as reasonablypredictive of PIV activity in humans (including, mice, hamsters, cottonrats, non-human primates including African green monkeys andchimpanzees), and humans, including seronegative and seropositiveinfants, children, juveniles, and adults. However, viruses and subviralparticles also can be designated that are highly defective forreplication in vivo.

In certain detailed aspects of the invention, the polynucleotidemolecule encoding the recombinant HPIV2 genome or antigenome encodes asequence of a wild-type HPIV2. Alternatively, the genome or antigenomemay bear one or more mutations from a biologically derived mutant HPIV2,or any combination of recombinantly-introduced mutation(s); includingone or more polynucleotide insertions, deletions, substitutions, orrearrangements that is/are selected to yield desired phenotypiceffect(s) in the recombinant virus.

Thus, the recombinant HPIV2 genome or antigenome may be engineeredaccording to the methods of the invention to incorporate arecombinantly-introduced restriction site marker, or a translationallysilent point mutation for handling or marking purposes. In otherembodiments, the polynucleotide molecule encoding the recombinant HPIV2genome or antigenome may incorporate one or morerecombinantly-introduced attenuating mutations. In exemplaryembodiments, the recombinant HPIV2 genome or antigenome incorporates oneor more recombinantly-introduced, temperature sensitive (ts) or hostrange (hr) attenuating (att) mutations.

Often, the recombinant HPIV2 genome or antigenome will incorporate oneor more attenuating mutation(s) identified in a heterologous mutant HPIVstrain (such as HPIV1 or HPIV3), or in another mutant nonsegmentednegative stranded RNA virus, for example RSV or murine PIV1 (MPIV1). Forexample, the recombinant HPIV2 genome or antigenome can be modified orconstructed to incorporate one or more mutation(s) corresponding tomutation(s) identified in a HPIV2, or a heterologous PIV such as thewell known candidate HPIV3 JS cp45. Useful mutations of HPIV3 JS cp45 oranother mutant virus can specify a change in a HPIV2 protein selectedfrom L, M, N, F, or FIN or in a HPIV2 extragenic sequence selected froma 3′ leader or N gene start sequence. Where the mutation relates to aparticular amino acid residue, the recombinant HPIV2 genome orantigenome will often incorporate multiple nucleotide changes in a codonspecifying the mutation to stabilize the modification against reversion.

In additional aspects of the invention, the recombinant HPIV2 genome orantigenome comprises an additional nucleotide modification specifying aphenotypic change selected from a change in growth characteristics,attenuation, temperature-sensitivity, cold-adaptation, plaque size,host-range restriction, or a change in immunogenicity. These additionalmodifications can alter one or more of the HPIV2 N, V, P, M, F, HNand/or L genes and/or a 3′ leader, 5′ trailer, a cis-acting sequencesuch as a gene start (GS) or gene end (GE) sequence, and/or intergenicregion within the HPIV2 genome or antigenome. For example, one or moreHPIV2 gene(s) can be deleted in whole or in part, or expression of thegene(s) can be reduced, ablated, or increased by a mutation in an RNAediting site, by a frameshift mutation, by a mutation that alters atranslation start site, by introduction of one or more stop codons in anopen reading frame (ORF) of the gene, or by a mutation in atranscription signal. In specific embodiments, the recombinant HPIV2genome or antigenome is modified by a partial or complete deletion ofthe HPIV2 V gene, or one or more nucleotide change(s) that reduces orablates expression of one or more HPIV2 genes yet yields a viable,replication competent, infectious viral construct. In other embodiments,the recombinant HPIV2 genome or antigenome is modified to encode anon-PIV molecule selected from a cytokine, a T-helper epitope, arestriction site marker, or a protein of a microbial pathogen capable ofeliciting an immune response in a mammalian host.

In yet additional aspects of the invention, the recombinant HPIV2 genomeor antigenome comprises a partial or complete HPIV2 “vector” genome orantigenome that is combined with one or more heterologous gene(s) orgenome segment(s) encoding one or more antigenic determinant(s) of oneor more heterologous pathogen(s) to form a chimeric HPN2 genome orantigenome. The heterologous gene(s) or genome segment(s) encoding theantigenic determinant(s) can be added as supernumerary gene(s) or genomesegment(s) adjacent to or within a noncoding region of the partial orcomplete HPIV2 vector genome or antigenome, or can be substituted forone or more counterpart gene(s) or genome segment(s) in a partial HPIV2vector genome or antigenome. The heterologous gene(s) or genomesegment(s) can include one or more heterologous coding sequences and/orone or more heterologous regulatory element(s) comprising an extragenic3′ leader or 5′ trailer region, a gene-start signal, gene-end signal,editing region, intergenic region, or a 3′ or 5′ non-coding region.

In more detailed embodiments, the heterologous pathogen is one or moreheterologous PIV(s) (e.g., HPIV1 and/or HPIV3) and the heterologousgene(s) or genome segment(s) encode(s) one or more PIV N, P, V, M, F, HNand/or L protein(s) or fragment(s) thereof. Thus, the antigenicdeterminant(s) may be selected from HPIV1 and HPIV3 HN and Fglycoproteins, and antigenic domains, fragments and epitopes thereof,that is/are added to or substituted within the partial or complete HPIV2genome or antigenome. In certain exemplary embodiments, genes encodingHN and F glycoproteins of HPIV3 or HPIV1 are substituted for counterpartHPIV2 HN and F genes in a partial HPIV2 vector genome or antigenome. Inmore detailed embodiments, the partial or complete HPIV2 genome orantigenome is modified to incorporate one or more gene(s) or genomesegment(s) encoding one or more antigenic determinant(s) of HPIV1, andone or more gene(s) or genome segment(s) encoding one or more antigenicdeterminant(s) of HPIV3, to yield a chimeric HPIV2 capable of elicitingan immune response against HPIV1 and HPIV3 in a, mammalian host. In thismanner, a plurality of heterologous genes or genome segments encodingantigenic determinants of multiple heterologous PIVs can be added to orincorporated within the partial or complete HPIV vector genome orantigenome.

In related embodiments of the invention, chimeric HPIV2 viruses areprovided wherein the vector genome is combined with one or moreheterologous antigenic determinant(s) of a heterologous pathogenselected from measles virus, subgroup A and subgroup B respiratorysyncytial viruses, mumps virus, human papilloma viruses, type I and type2 human immunodeficiency viruses, herpes simplex viruses,cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses,bunyaviruses, flaviviruses, alphaviruses, human metapneumoviruses, andinfluenza viruses. In exemplary aspects, the heterologous antigenicdeterminant(s) is/are selected from measles virus HA and F proteins,subgroup A or subgroup B respiratory syncytial virus F, G; SH and M2proteins, mumps virus HN and F proteins, human papilloma virus L1protein, type 1 or type 2 human immunodeficiency virus gp160 protein,herpes simplex virus and cytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ,gK, gL, and gM proteins, rabies virus G protein, Epstein Barr Virusgp350 protein, filovirus G protein, bunyavirus G protein, flavivirus preM, E, and NS1 proteins, human metapneuomovirus (HMPV) G and F protein,and alphavirus E protein, and antigenic domains, fragments and epitopesthereof. In certain specific embodiments, the heterologous pathogen ismeasles virus and the heterologous antigenic determinant(s) is/areselected from the measles virus HA and F proteins and antigenic domains,fragments and epitopes thereof. For example, a transcription unitcomprising an open reading frame (ORF) of a measles virus HA gene can beadded to or incorporated within a HPIV2 vector genome or antigenome toyield a chimeric candidate useful to immunize against measles and/orHPIV2 or another HPIV.

In additional embodiments, the partial or complete HPIV2 vector genomeor antigenome is modified to incorporate one or more supernumeraryheterologous gene(s) or genome segment(s) to form the chimeric HPIV2genome or antigenome. Typically, the supernumerary gene(s) or genomesegments(s) encode(s) one or more heterologous antigenic determinant(s),although non-coding inserts are also useful within recombinant, chimericHPIV2 of the invention. In exemplary embodiments, one or moresupernumerary heterologous gene(s) or genome segment(s) may be selectedfrom HPIV1 HN, HPIV2 F, HPIV3 HN, HPIV3 F, measles HA and F, HMPV G andF proteins, and/or RSV subgroup A or B G and F proteins. These and othersupernumerary heterologous gene(s) or genome segment(s) can be insertedat various sites within the recombinant genome or antigenome, forexample at a position 3′ to N, between the N/P, P/M, and/or HN/L genes,or at another intergenic junction or non-coding region of the HPIV2vector genome or antigenome.

In more detailed embodiments, the chimeric HPIV2 genome or antigenome isengineered to encode antigens from one, two, three or four pathogens.For example, the genome or anti genome may encode antigenic determinantsfrom one, two, three, four, or more different pathogens selected from aHPIV1, HPIV2, HPIV3, measles virus, respiratory syncytial virus, mumpsvirus, human papilloma virus, type 1 or type 2 human immunodeficiencyvirus, herpes simplex virus, cytomegalovirus, rabies virus, Epstein BarrVirus, filovirus, bunyavirus, flavivirus, alphavirus, humanmetapneumovirus, or influenza virus.

Where a gene or genome segment is added to or substituted within arecombinant HPIV2 genome or antigenome of the invention, it may be addedor substituted at a position corresponding to a wild-type gene orderposition of a counterpart gene or genome segment within the partial orcomplete HPIV2 genome or antigenome, which is often the case whenchimeric HPIV2 are generated by addition or substitution of aheterologous gene or genome segment into a partial or complete HPIV2vector genome or antigenome. Alternatively, the added or substituted(e.g., heterologous) gene or genome segment can be located at a positionthat is more promoter-proximal or promoter-distal compared to awild-type gene order position of a counterpart gene or genome segmentwithin the partial or complete HPIV2 background genome or antigenome.

In additional aspects of the invention, chimeric HPIV2 candidates areprovided wherein the HPIV2 vector genome or antigenome is modified toencode a chimeric glycoprotein incorporating one or more heterologousantigenic domains, fragments, or epitopes of a heterologous PIV, or of anon-PIV pathogen to form a chimeric genome or antigenome. In certainembodiments, the HPIV2 vector genome or antigenome is modified to encodea chimeric glycoprotein incorporating one or more antigenic domains,fragments, or epitopes from a second, antigenically distinct PIV to formthe chimeric genome or antigenome. Additional embodiments include achimeric HPIV2 wherein the genome or antigenome encodes a chimericglycoprotein having antigenic domains, fragments, or epitopes from twoor more HPIVs. In one example, the heterologous genome segment encodes aglycoprotein cytoplasmic, transmembrane or ectodomain which issubstituted for a corresponding glycoprotein domain in the HPIV2 vectorgenome or antigenome. In more specific embodiments, one or moreheterologous genome segment(s) of a second, antigenically distinct HPIVencoding one or more antigenic domains, fragments, or epitopes is/aresubstituted within a HPIV2 vector genome or antigenome to encode saidchimeric glycoprotein. For example, the one or more heterologous genomesegment(s) can be selected from ectodomains of HPIV1 and/or HPIV3 HNand/or F glycoproteins.

The chimeric HPIV2 candidates of the invention will typically bemodified as described above for non-chimeric HPIV2 recombinants, e.g.,by introduction of one or more attenuating mutations identified in aheterologous mutant PIV or other mutant nonsegmented negative strandedRNA virus. Thus, the HPIV2 genome or antigenome, or the chimeric HPIV2genome or antigenome, can be modified to incorporate one or more pointmutation(s), for example point mutations in one or more non-codingnucleotides or point mutations specifying an amino acid substitution,deletion or insertion, such as are identified in HPIV3 JS cp45.

In more detailed embodiments, the genome or antigenome of a chimeric ornon-chimeric HPIV2 is modified to incorporate one or any combination ofmutation(s) selected from mutations specifying previously identifiedamino acid substitution(s) in the L protein at a position correspondingto Tyr942, Leu992, and Thr1558 of HPIV3 JS cp45. Corresponding targetsof wild-type (wt) HPIV2 L for incorporation of these exemplary mutationsare Tyr948, Ala998, and Leu1566. In other embodiments, the recombinantHPIV2 genome or antigenome is modified to incorporate an attenuatingmutation at an amino acid position corresponding to an amino acidposition of an attenuating mutation identified in a heterologous, mutantnonsegmented negative stranded RNA virus, for example, respiratorysyncytial virus (RSV).

In yet additional detailed embodiments, the recombinant HPIV2 genome orantigenome is further modified to incorporate an additional nucleotidemodification specifying a phenotypic change selected from the following:a change in growth characteristics, attenuation,temperature-sensitivity, cold-adaptation, plaque size, host-rangerestriction, or immunogenicity. Such additional nucleotide modificationscan alter one or more ORFs, including but not limited to the HPIV1 N, P,V, M, F, HN and/or L ORFs and/or a 3′ leader, 5′ trailer, and/orintergenic region within the HPIV2 genome or antigenome. In exemplaryembodiments, the chimeric HPIV2 genome or antigenome is further modifiedsuch that one or more HPIV2 gene(s) is/are deleted in whole or in partor expression of the gene(s) is reduced, ablated, or increased by amutation in an RNA editing site, by a frameshift mutation, by a mutationthat alters a translation start site, by introduction of one or morestop codons in an open reading frame (ORF) of the gene, or by a mutationin a transcription signal. Often, the chimeric HPIV2 genome orantigenome will be engineered to incorporate a partial or completedeletion of the PIV V ORF or another non-essential gene or genomesegment, or one or more nucleotide change(s) that reduces, ablates, orincreases expression of one or more PIV genes. In other aspects, thechimeric HPIV2 genome or antigenome is modified to encode a non-PIVmolecule selected from a cytokine, a T-helper epitope, a restrictionsite marker, or a protein of a microbial pathogen capable of elicitingan immune response in a mammalian host.

In still other aspects of the invention, the recombinant HPIV2 genome orantigenome is recombinantly modified to form a chimeric HPIV2 genome orantigenome incorporating one or more heterologous genes or genomesegments from a non-human (e.g., bovine or SV5) PIV, to yield ahuman-non-human chimeric candidate having novel phenotypic properties,e.g., increased genetic stability, or altered attenuation,reactogenicity or growth in culture. Such recombinants may be producedby constructing a partial or complete HPIV2 vector genome or antigenomecombined with one or more heterologous genes or genome segments from anon-human PIV. For example, the partial or complete HPIV2 vector genomeor antigenome can be combined with one or more heterologous gene(s) orgenome segment(s) of a N, P, L, V, or M gene of a BPIV3 to form ahuman-bovine chimeric genome or antigenome and produce novel candidateshaving a host-range (hr) attenuation phenotype. In more detailedembodiments, a bovine PIV type 3 (BPIV3) N, M, L, V, or P open readingframe (ORF) or a genome segment thereof is substituted for a counterpartHPIV2 N, M, L, V, or P ORF or genome segment to form the chimericHPIV2-BPIV genome or antigenome. Alternatively, the PIV from which theheterologous gene(s) or genome segment(s) are donated to form thechimeric virus can be murine parainfluenza virus (MPIV), simian virus 5(SV5), SV41, Newcastle disease virus (NDV), or other animal PIV.

In further aspects of the invention, the recombinant HPIV2 genome orantigenome incorporates a polynucleotide insertion of between 150nucleotides (nts) and 4,000 nucleotides in length in a non-coding region(NCR) of the genome or antigenome or as a separate gene unit (GU). Therecombinant HPIV2 candidates comprising NCR and GU inserts replicateefficiently in vitro and typically exhibit an attenuated phenotype invivo. The polynucleotide insertion will typically lack a complete openreading frame (ORF) and will often specify an attenuated phenotype inthe recombinant HPIV2. The polynucleotide insert can be introduced intothe HPIV2 genome or antigenome in a reverse, non-sense orientationwhereby the insert does not encode protein. In more specificembodiments, the polynucleotide insert is approximately 2,000 nts, 3,000nts, or greater in length. In other embodiments, the polynucleotideinsertion adds a total length of foreign sequence to the recombinantHPIV2 genome or antigenome of 30% to 50% or greater compared to thewild-type HPIV2 genome length of approximately 15,600 nt (e.g., 15,654nt). In more detailed aspects, the polynucleotide insertion specifies anattenuation phenotype of the recombinant HPIV2 which exhibits at least a10- to 100-fold decrease in replication in the upper and/or lowerrespiratory tract.

In other embodiments of the invention polynucleotide molecules thatencode, or correspond, to a recombinant HPIV2 or chimeric HPIV2 genomeor antigenome as described above are provided. In additionalembodiments, polynucleotide expression vectors or constructs comprisinga polynucleotide encoding a recombinant HPIV2 or chimeric HPIV2 genomeor antigenome as described above and operably connected to expressionregulatory sequences (e.g., promotor and terminator sequences) to directexpression of the vector in suitable host cell or cell-free expressionsystem. In yet additional embodiments, a cell or cell-free expressionsystem (e.g., a cell-free lysate) is provided which incorporates anexpression vector comprising an isolated polynucleotide moleculeencoding a recombinant HPIV2 genome or antigenome, as described above,and optionally including an expression vector comprising one or moreisolated polynucleotide molecules encoding N, P, and L proteins of aPIV. One or more of the N, P, and L proteins may be expressed from HPIV2or from a heterologous PIV. Upon expression, the genome or antigenomeand N, P, and L proteins combine to produce an infectious HPIV particle,such as a viral or subviral particle. The isolated polynucleotidemolecules encoding the HPIV2 genome or antigenome and the one or moreisolated polynucleotide molecules encoding N, P, and L proteins of PIVcan be expressed by a single vector. Alternatively, the genome and oneor more of the N, P, and L proteins can be incorporated into two or moreseparate vectors.

The recombinant HPIV2 viruses of the invention are useful in variouscompositions to generate a desired immune response against one or morePIVs, or against PIV and one or more non-PIV pathogen(s), in a hostsusceptible to infection therefrom. Recombinant HPIV2 as disclosedherein are capable of eliciting a mono- or poly-specific immune responsein an infected mammalian host, yet are sufficiently attenuated so as tonot cause unacceptable symptoms of disease in the immunized host. Theattenuated viruses, including subviral particles, may be present in acell culture supernatant, isolated from the culture, or partially orcompletely purified. The virus may also be lyophilized, and can becombined with a variety of other components for storage or delivery to ahost, as desired.

The invention further provides novel immunogenic compositions comprisinga physiologically acceptable carrier and/or adjuvant and an isolatedattenuated recombinant HPIV2 virus as described above. In preferredembodiments, the immunogenic composition is comprised of a recombinantHPIV2 having at least one, and preferably two or more attenuatingmutations or other nucleotide modifications that specify a suitablebalance of attenuation and immunogenicity, and optionally additionalphenotypic characteristics. The immunogenic composition can beformulated in a dose of 10³ to 10⁷ PFU of attenuated virus. Theimmunogenic composition may comprise attenuated recombinant HPIV2 thatelicits an immune response against a single PIV strain or againstmultiple PIV strains or serotypes or other pathogens such as RSV and/orHMPV. In this regard, recombinant HPIV2 can be combined in formulationswith other PIV strains, or with other candidate viruses such as a liveattenuated RSV.

In related aspects, the invention provides a method for stimulating theimmune system of an individual to elicit an immune response against oneor more PIVs, or against PIV and a non-PIV pathogen, in a mammaliansubject. The method comprises administering a formulation of animmunologically sufficient amount a recombinant HPIV2 in aphysiologically acceptable carrier and/or adjuvant. In one embodiment,the immunogenic composition is comprised of a recombinant HPIV2 havingat least one, and preferably two or more attenuating mutations or othernucleotide modifications specifying a desired phenotype and/or level ofattenuation as described above. The immunogenic composition can beformulated in a dose of 10³ to 10⁷ PFU of attenuated virus. Theimmunogenic composition may comprise a recombinant HPIV2 that elicits animmune response against a single PIV, against multiple PIVs, e.g., HPIV2and HPIV3, or against one or more PIV(s) and a non-PIV pathogen such asmeasles or RSV.

In this context, recombinant HPIV2 viruses of the invention can elicit amonospecific immune response or a polyspecific immune response againstmultiple PIVs, or against one or more PIV(s) and a non-PIV pathogen.Alternatively, recombinant HPIV2 having different immunogeniccharacteristics can be combined in a mixture or administered separatelyin a coordinated treatment protocol to elicit more effective immuneresponse against one PIV, against multiple PIVs, or against one or morePIV(s) and a non-PIV pathogen such as measles or RSV. Typically, theimmunogenic compositions of the invention are administered to the upperrespiratory tract, e.g., by spray, droplet or aerosol.

In other aspects of the invention, novel recombinant HPIV1 viruses andrelated compositions and methods are also provided. These HPIV1recombinant viruses are useful in combination with the HPIV2compositions and methods described herein, and provide additionalexemplary mutations and other modifications for incorporation intorecombinant HPIV1 and HPIV2 for use within immunogenic compositions andmethods. As in the case of HPIV2, these embodiments of the invention arebased on production of an infectious, self-replicating, recombinanthuman parainfluenza virus type 1 (HPIV1) from one or more isolatedpolynucleotide molecules encoding the virus. Foundational aspects ofthese embodiments are described, for example, in U.S. patent applicationSer. No. 10/302,547, filed by Murphy et al. on Nov. 21, 2002 and in thecorresponding PCT Publication Number WO 03/043587 A2, published on May30, 2003 (each incorporated herein by reference).

In certain embodiments, the polynucleotide molecule encoding therecombinant HPIV1 genome or antigenome bears one or more mutations froma biologically derived mutant HPIV1, or any combination ofrecombinantly-introduced mutation(s); including one or morepolynucleotide insertions, deletions, substitutions, or rearrangementsthat is/are selected to yield desired phenotypic effect(s) in therecombinant virus. In exemplary embodiments, the recombinant HPIV1genome or antigenome incorporates one or more recombinantly-introduced,temperature sensitive (ts) or host range (hr) attenuating (att)mutations. Often, the recombinant HPIV1 genome or antigenome willincorporate one or more attenuating mutation(s) identified in abiologically derived mutant PIV strain, or in another mutantnonsegmented negative stranded RNA virus, for example RSV or murine PIV(MPIV). For example, the recombinant HPIV1 genome or antigenome can bemodified or constructed to incorporate one or more mutation(s)corresponding to mutation(s) identified in a HPIV1, or a heterologousPIV such as the well known immunogenic composition candidate HPIV3 JScp45. Useful mutations of HPIV3 JS cp45 or another mutant virus canspecify a change in a HPIV1 protein selected from L, M, N, C, F, or FINor in a HPIV1 extragenic sequence selected from a 3′ leader or N genestart sequence. Where the mutation relates to a particular amino acidresidue, the recombinant HPIV1 genome or antigenome will oftenincorporate multiple nucleotide changes in a codon specifying themutation to stabilize the modification against reversion.

In additional aspects of the invention, the recombinant HPIV1 genome orantigenome comprises an additional nucleotide modification specifying aphenotypic change selected from a change in growth characteristics,attenuation, temperature-sensitivity, cold-adaptation, plaque size,host-range restriction, or a change in immunogenicity. These additionalmodifications can alter one or more of the HPIV1 N, P, C, C′, Y1, Y2, M,F, HN and/or L genes and/or a 3′ leader, 5′ trailer, a cis-actingsequence such as a gene start (GS) or gene end (GE) sequence, and/orintergenic region within the HPIV1 genome or antigenome. For example,one or more HPIV1 gene(s) can be deleted in whole or in part, orexpression of the gene(s) can be reduced or ablated by a mutation in anRNA editing site, by a frameshift mutation, by a mutation that alters atranslation start site, by introduction of one or more stop codons in anopen reading frame (ORF) of the gene, or by a mutation in atranscription signal. In specific embodiments, the recombinant HPIV1genome or antigenome is modified by a partial or complete deletion ofone or more C, C′, Y1, and/or Y2 ORF(s) or other auxiliary gene, or oneor more nucleotide change(s) that reduces or ablates expression of oneor more of the C, C′, Y1, and/or Y2 ORF(s) or other auxiliary gene. Inother embodiments, the recombinant HPIV1 genome or antigenome ismodified to encode a non-PIV molecule selected from a cytokine, aT-helper epitope, a restriction site marker, or a protein of a microbialpathogen capable of eliciting an immune response in a mammalian host.

In yet additional aspects of the invention, the recombinant HPIV1 genomeor antigenome comprises a partial or complete HPIV1 “vector” genome orantigenome that is combined with one or more heterologous gene(s) orgenome segment(s) encoding one or more antigenic determinant(s) of oneor more heterologous pathogen(s) to form a chimeric HPIV1 genome orantigenome. The heterologous gene(s) or genome segment(s) encoding theantigenic determinant(s) can be added as supernumerary gene(s) or genomesegment(s) adjacent to or within a noncoding region of the partial orcomplete HPIV1 vector genome or antigenome, or can be substituted forone or more counterpart gene(s) or genome segment(s) in a partial HPIV1vector genome or antigenome. The heterologous gene(s) or genomesegment(s) can include one or more heterologous coding sequences and/orone or more heterologous regulatory element(s) comprising an extragenic3′ leader or 5′ trailer region, a gene-start signal, gene-end signal,editing region, intergenic region, or a 3′ or 5′ non-coding region.

In related embodiments of the invention, chimeric HPIV1 viruses areprovided wherein the vector genome is combined with one or moreheterologous antigenic determinant(s) of a heterologous pathogenselected from measles virus, subgroup A and subgroup B respiratorysyncytial viruses, mumps virus, human papilloma viruses, type I and type2 human immunodeficiency viruses, herpes simplex viruses,cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses,bunyaviruses, flaviviruses, alphaviruses, human metapneumoviruses, andinfluenza viruses. In exemplary aspects, the heterologous antigenicdeterminant(s) is/are selected from measles virus HA and F proteins,subgroup A or subgroup B respiratory syncytial virus F, G, SH and M2proteins, mumps virus HN and F proteins, human papilloma virus LIprotein, type 1 or type 2 human immunodeficiency virus gp160 protein,herpes simplex virus and cytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ,gK, gL, and gM proteins, rabies virus G protein, Epstein Barr Virusgp350 protein, filovirus G protein, bunyavirus G protein, flavivirus preM, E, and NS1 proteins, human metapneuomovirus G and F protein, andalphavirus E protein, and antigenic domains, fragments and epitopesthereof.

Modifications useful within rHPIV1 viruses of the invention can besimilarly incorporated into rHPIV2 viruses as described herein, andlikewise modifications useful within rHPIV2 viruses of the invention canbe incorporated into rHPIV1 for development of yet additionalimmunogenic compositions and methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (Panels A-E) illustrates various mutations identified in aheterologous negative stranded RNA virus that can be incorporated intorecombinant HPIV2 candidates of the invention to yield attenuation orother desired phenotypic changes. Partial amino acid sequence alignments(SEQ ID NOS 1-16, respectively in order of appearance) are providedbetween HPIV2 wild-type (wt), HPIV3 wt, HPIV1 wt, or BPIV3 wt forregions of the indicated protein that contain known attenuatingmutations. Based on these and similar comparisons, mutations areidentified in a heterologous PIV or non-PIV virus for transfer into arecombinant HPIV2 of the invention. The amino acid position andsubstitution previously shown to confer a phenotypic change in theheterologous virus is indicated above each sequence alignment and wildtype assignment is in bold font in the sequence alignment. Thecorresponding amino acid position in HPIV2 is indicated after the HPIV2sequence. Amino acid positions that are conserved between all species ofL protein in the alignment are underlined, representing additionaltargets for mutagenesis.

FIG. 2 illustrates modification of HPIV2 for use as a vector of otherviral antigens. Panel A: Diagram (not to scale) of the HPIV2 genomeillustrating the placement of a unique promoter-proximal Not Irestriction site. The location of HPIV2/V94 nts 56-160 in the completegenome diagram is indicated with a box, and the sequence is shown above(SEQ ID NO: 17). The sequence was modified from wild-type to contain aNot I restriction site (GCGGCCGC) one nucleotide prior to thetranslational start codon of the HPIV2 N gene (ATG, HPIV2/V94 nts158-160). The Not I site was introduced by changing the HPIV2 sequenceAGGTTCAA (HPIV2/V94 nts 149-156) to GCGGCCGC (Not I recognitionsequence). The position of two potential N gene-start signals isindicated as shaded areas. The promoter element (open box) is a sequencethat has been demonstrated to be important for viral replication andtranscription in other Paramyxoviruses. Panel B: Insertion of the HPIV1HN or RSV (subgroup A) G open reading frame as a supernumerary gene inthe Not I site (SEQ ID NOS 18, 19, 17, 18, 20, and 17, respectively inorder of appearance). A gene-start, intergenic, and gene-end sequenceidentical to that found between the HPIV2 N and P genes (HPIV2/V94 nts1909-1938) is inserted after each supernumerary ORF. These signals serveto terminate transcription of the foreign ORF and start transcription ofthe downstream HPIV2 N gene, respectively. Each supernumerary genecassette will be generated using PCR with a sense oligo that willinclude a Not I restriction site and an antisense oligo that containsgene-end (GE) and gene-start (GS) sequences that will be used toterminate transcription for the inserted gene and promote transcriptionfor the HPIV2 N gene, respectively. A unique BstEII site is alsoincluded which will allow for the optional insertion of a secondsupernumerary ORF. The entire sequence is modified as necessary toconform to the rule of six by adjusting the length (n) of the sequencein the positions indicated by the arrow. The bottom section for eachvirus details the sequence of the HPIV2 backbone where the ORFs are tobe inserted. This strategy can be used to engineer other uniquerestriction sites at any one of the gene junctions to allow for theinsertion of multiple foreign genes.

FIG. 3 provides a diagram (not to scale) of the assembled cDNA clone,pFLC HPIV2/V94, that yields the antigenomic RNA of HPIV2/V94 whentranscribed by T7 RNA polymerase. The 6 overlapping PCR fragments usedto assemble the full-length antigenomic cDNA are shown (A-F).Restriction sites along with their nucleotide positions that were usedto assemble the clone are shown above the boxed diagram of the viralgenome. The T7 polymerase promoter (T7) and two non-viral G residuesthat enhance transcription flank the upstream end of the antigenome, andthe hepatitis delta virus ribozyme sequence (A) flanks the downstreamend. The relative position of each HPIV2 ORF is shown. The nucleotidechanges from the consensus sequence of biologically derived HPIV2/V94are indicated (*) and the resulting amino acid change in the protein isshown.

FIG. 4 provides multi-step growth curves of HPIV2s. LLC-MK2 monolayerswere infected in triplicate with the indicated HPIV2 at an m.o.i. of0.01 at 32° C. Aliquots of the medium supernatants were harvested at24-hour intervals and were assayed at 32° C. for virus titer. Virustiters are expressed as mean login TCID₅₀/ml±standard error. Panel A:Viruses were grown and titered in the absence of trypsin. Panel B:Viruses were grown with added porcine trypsin (5 ug/ml), and virus wasquantified on LLC-MK2 cells with added trypsin.

FIG. 5 illustrates in vivo characterization of rHPIV2N94. Replication ofthe recombinant virus was studied in hamsters, an accepted animal modelsystem for predicting growth and attenuation characteristics in humanhosts. Hamsters were inoculated i.n. with 10⁶ TCID₅₀ of the indicatedvirus. Nasal tubinates and lung tissues from six animals from each groupwere harvested on days three, four, and five post-infection. Viruspresent in tissues was quantified by serial dilution on LLC-MK2monolayers at 32° C. r_(A)HPIV2/V94 and r_(B)HPIV2/V94 are preparationsof HPIV2 derived from separate transfections.

FIG. 6 provides a comparison of selected regions of the nucleotidesequence of the HPIV2 Toshiba and Greer strains (SEQ ID NOS 21-40,respectively in order of appearance). The Toshiba and Greer sequenceswere aligned using the BESTFIT alignment program (Wisconsin PackageVersion 10.2, Genetics Computer Group (GCG), Madison, Wis.). Missing ntsare indicated by (.). Ten regions were identified (A-J) that likelyrepresent sequence errors in the reported Toshiba strain sequence. Thesesequences (antigenomic sense) are found in (A), the Toshiba strain Ngene start signal sequence missing 1-nt, (B) the Toshiba strain N ORF,missing codon-195 (Arg), (C) the Toshiba strain N gene end (containing 1extra nt) and P gene start signals (missing 1-nt), (D) P ORF (missing 1nt), (E) P ORF (containing 1 extra nt) resulting in missense of P aa316319 (changes GSDM (SEQ ID NO: 67) to QVIL (SEQ ID NO: 68)), (F) HN 3′NCR (missing 1 nt), (G) L ORF (missing codon-378 (Ala)), (H) L ORF(missing codon-741 (Pro)), (1-J) L ORF (containing 3 extra nts,resulting in missense of L polymerase aa 1735-1743 (changing TLTKFDLSL(SEQ ID NO: 69) to NSNKVRFIPF (SEQ ID NO: 70)).

FIG. 7 illustrates the structure of nucleotide inserts used to makeHPIV2 antigenomic cDNAs that do not conform to the rule of six. Panel A.The wt nt sequence (SEQ ID NO: 41) around the EcoRV restriction sitenear the end of the L ORF is shown. Panel B. The sequences of the sixantigenomic-sense oligonucleotides (SEQ ID NO: 42-47, respectively inorder of appearance) inserted at the EcoRV restriction site spanningHPIV2 nt 15554-15559 is shown. Oligonucleotide duplexes were insertedbetween nt 15556 and 15557. Each oligonucleotide duplex contains asilent ATC to att mutation that destroys the EcoRV site and recreatesthe last 11 codons of the L ORF, and 12 HPIV2 nt including the TGA stopcodon (bold) followed by 0-5 additional nt. The designation of therecombinant viruses generated from the cDNAs is indicated to the left:the virus designated rHPIV2N94(+6) corresponds to the rule of six, whilethe others contain the indicated number of additional nt (+1 to +5). Thelength of each oligonucleotide insert is shown on the right, with therule of six length and the total length of the antigenomic cDNAindicated in parenthesis.

FIG. 8 illustrates nucleotide insertions and deletions detected in thegenomic RNA of recombinant HPIV2s derived from cDNAs that do not conformto the rule of six (sequences are in antigenomic sense). Panel A. Fourrecombinant viruses were produced from cDNAs that did not conform to therule of six, rHPIV2N94(+3), rHPIV2/V94(+4) (SEQ ID NO: 50),r_(A)HPIV2/V94(+5) (SEQ ID NO: 51), and r_(B)HPIV2N94(+5) (SEQ ID NO:52), contained nt insertions that resulted in a polyhexameric genomelength. Two insertions containing a total of 3 nt were identified in theHN 5′ and 3′ noncoding regions (5′ NCR (SEQ ID NO: 48) and 3′ NCR (SEQID NO: 49)) of rHPIV2N94(+3). A 2-nt insertion within the intergenic(IG) region between the HN and L genes was identified in rHPIV2N94(+4).A 1-nt insertion was found in each clone of r_(A)HPIV2/V94(+5) andr_(B)HPIV2/V94(+5), in one case at the end of the HN GE signal and inthe other in the HN 3′ NCR. Panel B. Two recombinant viruses producedfrom cDNAs that did not conform to the rule of six, rHPIV2/V94(+2) (SEQID NO:53) and rHPIV2N94(+1) (SEQ ID NO: 56), contained nt deletions thatresulted in a polyhexameric genome length. rHPIV2N94(+2) was found tohave a 2-nt deletion (underlined) within the IG region between the HNand L genes. rHPIV2N94(+1) was found to have a 1-nt deletion(underlined) near the end of the L polymerase ORF (SEQ ID NO: 54). Thisresulted in a frame shift in the L coding sequence that deleted the last13 amino acids (SEQ ID NO: 55) of L and replaced them with an unrelatedsequence of 21 amino acids (SEQ ID NO: 54), as shown. The nt sequence(antigenomic sense) in the region of the insertions is shown. Theinserted nt are shown and the site of insertion is indicated by an arrowpointing downward. Deleted nt are shown and the location of the deletionis indicated by an arrow pointing upward. The HN 5′ or 3′ non-codingregion (NCR), transcription gene end (GE) and gene-start (GS) (in boldtype) and intergenic regions (IG) between the HPIV2 HN and L ORFs areindicated. The L polymerase translation initiation codon (ATG) andtranslation termination codon (TGA) are underlined. The single letteramino acid designation is shown below the nt sequence for the wt and amutant version of the HPIV2 L polymerase.

FIGS. 9A-F provide a nucleotide sequence for the HPIV2/V94 strain (SEQID NO: 58).

FIGS. 10A-F provide a nucleotide sequence for the HPIV2/V98 strain (SEQID NO: 59).

FIGS. 11A-F provide a nucleotide sequence for the HPIV2 Greer strain(SEQ ID NO: 60).

FIG. 12 illustrates the level of virus shedding in the upper and lowerrespiratory tract of African green monkeys infected with either thebiologically derived HPIV2 or a recombinant HPIV2 containing a two aminoacid deletion at positions 1764 and 1765 in the L polymerase protein.This “imported” mutation was initially identified in a recombinanthuman-bovine chimeric HPIV3 (designated rHPIV3-L_(B)), and thecorresponding target site for the mutation in HPIV2 is at residue 1764.Substitutions and deletions at this target site are contemplated togenerate attenuated HPIV2 constructs. In this example, a two residuedeletion was engineered to delete the subject target residue along witha second residue to conform the cDNA construct to the “rule of six”,although other 1764 deletion or substitution constructs can be readilygenerated without altering the 1765 residue. The lower limit ofdetection in the subject assay is indicated.

FIG. 13 illustrates multicycle replication of rHPIV2-FRSV and therecombinant parent virus, rHPIV2/V94, in simian LLC-MK2 cells.Triplicate monolayer cultures were infected at an input m.o.i. of 0.01TCID₅₀ per cell with the wild type recombinant HPIV2 and the recombinantHPIV2 vector expressing the RSV fusion protein. The virus titers areshown as mean log₁₀ TCID₅₀/ml of triplicate samples.

FIG. 14 illustrates modification of a recombinant HPIV1 of the inventionfor use as a vector for heterologous protective antigens of differentPIV and non-PIV pathogens according to the invention. Panel A provides adiagram of the HPIV1 genome that has been modified from wild-type tocontain an Mlu I restriction site one nucleotide prior to thetranslational start codon of the N protein (starting at HPIV1 nt 113),or a Not I restriction site between the P and M ORFs, within the P gene3′ non-coding region (starting at HPIV1 nt 3609). Gene-start andgene-stop signals for each HPIV1 gene are shaded in gray and black,respectively. In panel B, the area enclosed by the rectangle around theMlu I site is expanded and illustrates the insertion of the HMPV strainCAN83 F protein ORF (the complete F ORF is 1620 nt in length and encodesa 539 as polypeptide. The length of the entire inserted supernumerarygene unit sequence is 1656 nt). For the recombinant virus that isillustrated in panel B, rHPIV1-F₈₃, the top sequence shows the insertthat is generated using PCR with a sense oligo that includes an Mlu Irestriction site (SEQ ID NO: 69) and an antisense oligo (SEQ ID NO: 62)that contains gene-stop and gene-start sequences that are used toterminate transcription for the inserted gene and promote transcriptionfor the N gene, respectively. Additional nucleotides (indicated by R6)are inserted where necessary to conform the entire inserted sequence tothe rule of six, and to maintain the HPIV1 gene start signal sequencephasing (Kolakofsky et al., J. Virol. 72: 891-899, 1998, incorporatedherein by reference). The bottom section in the panel details thesequence of the HPIV1 backbone where the ORF is inserted (SEQ ID NO:63). The naturally occurring gene-start sequence is boxed. The promoterelement is a sequence that has been demonstrated to be important forviral replication and transcription. In panel C, the area enclosed bythe rectangle around the Not I site is expanded and illustrates theinsertion of the HMPV CAN83 strain G protein ORF from the CAN83 strainof HMPV (the complete G ORF is 660 nt in length and encodes a 179 aapolypeptide. The length of the entire inserted supernumerary gene unitsequence is 702 nt). For the recombinant virus that is illustrated inpanel C, rHPIV1-G₈₃, the top sequence shows the insert that is generatedusing PCR with a sense (SEQ ID NO: 64) oligo that includes a Not Irestriction site, gene-stop and gene-start sequences that are used toterminate transcription for the upstream P gene and promotetranscription for the inserted supernumerary gene unit, respectively,and an antisense oligo that contains a NotI site. Additional nucleotides(indicated by R6) are inserted where necessary to conform the entireinserted sequence to the rule of six, and to maintain the HPIV1 genestart signal sequence phasing. The bottom section in the panel detailsthe sequence of the HPIV1 backbone at the P-M junction where the ORF isinserted. The naturally occurring gene-end and gene start sequences areboxed. This strategy can be used to engineer other unique restrictionsites at any one of the gene junctions or 3′ or 5′ portions of thegenome or antigenome to allow for the insertion of foreign genes, asdesired.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The instant invention provides methods and compositions for theproduction and use of novel human parainfluenza virus type 2 (HPIV2)candidates. The recombinant HPIV2 viruses of the invention areinfectious and immunogenic in humans and other mammals and are usefulfor generating immune responses against one or more PIVs, for exampleagainst one or more human PIVs (HPIVs). In additional embodiments,chimeric HPIV2 viruses are provided that elicit an immune responseagainst a selected PIV and one or more additional pathogens, for exampleagainst multiple HPIVs or against a HPIV and a non-PIV virus such asrespiratory syncytial virus (RSV), human metapneumovirus, or measlesvirus. The immune response elicited can involve either or both humoraland/or cell mediated responses. Preferably, recombinant HPIV2 viruses ofthe invention are attenuated to yield a desired balance of attenuationand immunogenicity. The invention thus provides novel methods fordesigning and producing attenuated, HPIV2 viruses that are useful asagents for eliciting a desired immune response against HPIV2 and otherpathogens. An important feature of the invention is that it provides forthe production, with high frequency, of recombinant PIVs having adefined genome sequence and predictable characteristics.

Exemplary recombinant HPIV2 viruses of the invention incorporate arecombinant HPIV2 genome or antigenome, as well as a PIV majornucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), and a largepolymerase protein (L). The N, P, and L proteins may be HPIV2 proteins,or one or more of the N, P, and L proteins may be of a different HPIV,for example HPIV1 or HPIV3. Additional PIV proteins may be included invarious combinations to provide a range of infectious viruses, definedherein to include subviral particles lacking one or more non-essentialviral components and complete viruses having all native viralcomponents, as well as viruses containing supernumerary proteins,antigenic determinants or other additional components.

As set forth in the examples below, a complete consensus sequence wasdetermined herein for the genomic RNA of a human parainfluenza virustype 2 (HPIV2) strain Vanderbilt/1994 (V94), a clinical isolate that wasoriginally isolated from an infected, one year-old infant. The sequencethus identified was used to generate a full-length antigenomic cDNA andto recover a recombinant wild type HPIV2 (rHPIV2).

The replication of rHPIV2 in vitro and in the respiratory tract ofhamsters was similar to that of its biologically derived parent virus.The biological properties of rHPIV2 in vitro and in vivo demonstratesthat the rHPIV2 sequence corresponds to a wild type virus. This is acritical finding, since it demonstrates that the recombinant HPIV2sequence is that of an authentic wild-type virus. This rHPIV2 thereforeserves as a novel substrate for recombinant introduction of attenuatingmutations for the generation of live-attenuated HPIV2 and other PIVcandidates.

The Paramyxovirinae subfamily of the Paramyxoviridae family of virusesincludes human parainfluenza virus types 1, 2, 3, 4A and 4B (HPIV1,HPIV2, HPIV3, HPIV4A, and HPIV4B, respectively). HPIV1, HPIV3, MPIV1,and bovine PIV3 (BPIV3) are classified together in the genusRespirovirus, whereas HPIV2 and HPIV4 are more distantly related and areclassified in the genus Rubulavirus. MPIV1, simian virus 5 (SV5), andBPIV3 are animal counterparts of HPIV1, HPIV2, and HPIV3, respectively(Chanock et al., in Parainfluenza Viruses, Knipe et al. (Eds.), pp.1341-1379, Lippincott Williams & Wilkins, Philadelphia, 2001,incorporated herein by reference).

The human PIVs have a similar genomic organization, although significantdifferences occur in the P gene (Chanock et al., in ParainfluenzaViruses, Knipe et at. (eds.), pp. 1341-1379, Lippincott Williams &Wilkins, Philadelphia, 2001; Lamb et al., in Paramyxoviridae: Theviruses and their replication, Knipe et al. (eds.), pp. 1305-1340,Lippincott Williams & Wilkins, Philadelphia, 2001, each incorporatedherein by reference). The 3′ end of genomic RNA and its full-length,positive-sense replicative intermediate antigenomic RNA contain promoterelements that direct transcription and replication. Thenucleocapsid-associated proteins are composed of the nucleocapsidprotein (N), the phosphoprotein (P), and the large polymerase (L). Theinternal matrix protein (M) and the major antigenic determinants, thefusion glycoprotein (F) and hemagglutinin-neuraminidase glycoprotein(HN) are the envelope-associated proteins. The gene order is N, V/P, M,F, HN, and L.

With the exception of the P gene, each HPIV2 gene contains a single ORFand encodes a single viral protein. The P gene of the Paramyxovirinaesubfamily variably encodes a number of proteins that are generated fromalternative open reading frames (ORFs), by the use of alternativetranslational start sites within the same ORF, by an RNA polymeraseediting mechanism, by ribosomal shunting, or through ribosomal frameshifting (Lamb et al., in Paramyxoviridae: The viruses and theirreplication, Knipe et al. (Eds.), pp. 1305-1340, Lippincott Williams &Wilkins, Philadelphia, 2001; Liston et at., J Virol 69:6742-6750, 1995;Latorre et al., Mol. Cell. Biol. 18:5021-5031, 1998, incorporated hereinby reference). For example, the MPIV1 P gene expresses eight proteins.Four of these, C, C′, Y1, and Y2, are expressed by translationalinitiation at four different codons within the C ORF that is present ina +1 reading frame relative to the P ORF (Curran et al., Embo J.7:245-251, 1988, Dillon et al., J. Virol. 63:974-977, 1989; Curran etal., Virology 189:647-656, 1989, each, incorporated herein byreference). The HPIV2 P gene encodes the P protein and one additionalprotein, V. The V protein does not appear to be absolutely necessary forHPIV2 replication in cell culture. HPIV1 encodes a P protein but doesnot appear to encode a V protein, based on the lack of a homologous RNAediting site and the presence of a relict V coding sequence that isinterrupted by 9-11 stop codons (Matsuoka et al., J. Virol.65:3406-3410, 1991; Rochat et al., Virus Res. 24:137-144, 1992,incorporated herein by reference).

Infectious recombinant HPIV2 viruses according to the invention areproduced by a recombinant coexpression system that permits introductionof defined changes into the recombinant HPIV2 and provides for thegeneration, with high frequency and fidelity, of HPIV2 having a definedgenome sequence. These modifications are useful in a wide variety ofapplications, including the development of live attenuated viral strainsbearing predetermined, defined attenuating mutations. Infectious PIV ofthe invention are typically produced by intracellular or cell-freecoexpression of one or more isolated polynucleotide molecules thatencode the HPIV2 genome or antigenome RNA, together with one or morepolynucleotides encoding the viral proteins desired, or at leastnecessary, to generate a transcribing, replicating nucleocapsid.

cDNAs encoding a HPIV2 genome or antigenome are constructed forintracellular or in vitro coexpression with the selected viral proteinsto form infectious PIV. By “HPIV2 antigenome” is meant an isolatedpositive-sense polynucleotide molecule which serves as a template forsynthesis of progeny HPIV2 genome. Preferably a cDNA is constructedwhich is a positive-sense version of the HPIV2 genome corresponding tothe replicative intermediate RNA, or antigenome, so as to minimize thepossibility of hybridizing with positive-sense transcripts ofcomplementing sequences encoding proteins necessary to generate atranscribing, replicating nucleocapsid.

In some embodiments of the invention the genome or antigenome of arecombinant HPIV2 (rHPIV2) need only contain those genes or portionsthereof necessary to render the viral or subviral particles encodedthereby infectious. Further, the genes or portions thereof may beprovided by more than one polynucleotide molecule, i.e, a gene may beprovided by complementation or the like from a separate nucleotidemolecule. In other embodiments, the PIV genome or antigenome encodes allfunctions necessary for viral growth, replication, and infection withoutthe participation of a helper virus or viral function provided by aplasmid or helper cell line.

By “recombinant HPIV2” is meant a HPIV2 or HPIV2-like viral or subviralparticle derived directly or indirectly from a recombinant expressionsystem or propagated from virus or subviral particles producedtherefrom. The recombinant expression system will employ a recombinantexpression vector which comprises an operably linked transcriptionalunit comprising an assembly of at least a genetic element or elementshaving a regulatory role in PIV gene expression, for example, apromoter, a structural or coding sequence which is transcribed into PIVRNA, and appropriate transcription initiation and termination sequences.

To produce infectious HPIV2 from a cDNA-expressed HPIV2 genome orantigenome, the genome or antigenome is coexpressed with those PIV(HPIV2 or heterologous PIV) proteins necessary to produce a nucleocapsidcapable of RNA replication, and render progeny nucleocapsids competentfor both RNA replication and transcription. Transcription by the genomenucleocapsid provides the other PIV proteins and initiates a productiveinfection. Alternatively, additional PIV proteins needed for aproductive infection can be supplied by coexpression.

In certain embodiments of the invention, complementing sequencesencoding proteins necessary to generate a transcribing, replicatingHPIV2 nucleocapsid are provided by one or more helper viruses. Suchhelper viruses can be wild type or mutant. Preferably, the helper viruscan be distinguished phenotypically from the virus encoded by the HPIV2cDNA. For example, it may be desirable to provide monoclonal antibodiesthat react immunologically with the helper virus but not the virusencoded by the HPIV2 cDNA. Such antibodies can be neutralizingantibodies. In some embodiments, the antibodies can be used in affinitychromatography to separate the helper virus from the recombinant virus.To aid the procurement of such antibodies, mutations can be introducedinto the HPIV2 cDNA to provide antigenic diversity from the helpervirus, such as in the HN or F glycoprotein genes.

Expression of the HPIV2 genome or antigenome and proteins fromtransfected plasmids can be achieved, for example, by each cDNA beingunder the control of a selected promoter (e.g., for T7 RNA polymerase),which in turn is supplied by infection, transfection or transductionwith a suitable expression system (e.g., for the T7 RNA polymerase, suchas a vaccinia virus MVA strain recombinant which expresses the T7 RNApolymerase, as described by Wyatt et al., Virology 210:202-205, 1995,incorporated herein by reference). The viral proteins, and/or T7 RNApolymerase, can also be provided by transformed mammalian cells or bytransfection of preformed mRNA or protein.

A HPIV2 genome or antigenome may be constructed for use in the presentinvention by, e.g., assembling cloned cDNA segments, representing inaggregate the complete genome or antigenome, by polymerase chainreaction or the like (PCR; described in, e.g., U.S. Pat. Nos. 4,683,195and 4,683,202, and PCR Protocols: A Guide to Methods and Applications,Innis et al., eds., Academic Press, San Diego, 1990, each incorporatedherein by reference) of reverse-transcribed copies of HPIV2 mRNA orgenome RNA. For example, a first construct may be generated whichcomprises cDNAs containing the left hand end of the antigenome, spanningfrom an appropriate promoter (e.g., T7 RNA polymerase promoter) andassembled in an appropriate expression vector, such as a plasmid,cosmid, phage, or DNA virus vector. The vector may be modified bymutagenesis and/or insertion of a synthetic polylinker containing uniquerestriction sites designed to facilitate assembly. For ease ofpreparation the N, P, L and other desired PIV proteins can be assembledin one or more separate vectors. The right hand end of the antigenomeplasmid may contain additional sequences as desired, such as a flankingribozyme and single or tandem T7 transcriptional terminators. Theribozyme can be hammerhead type, which would yield a 3′ end containing asingle nonviral nucleotide, or can be any of the other suitableribozymes such as that of hepatitis delta virus (Perrotta et al., Nature350:434-436, 1991, incorporated herein by reference) that would yield a3′ end free of non-PIV nucleotides.

Alternative means to construct cDNA encoding the HPIV2 genome orantigenome include reverse transcription-PCR using improved PCRconditions (e.g., as described in Cheng et al., Proc. Natl. Acad. Sci.USA 91:5695-5699, 1994, incorporated herein by reference) to reduce thenumber of subunit cDNA components to as few as one or two pieces. Inother embodiments different promoters can be used (e.g., T3, SPQ ordifferent ribozymes, such as that of a hammerhead variety. Different DNAvectors (e.g., cosmids) can be used for propagation to betteraccommodate the larger size genome or antigenome.

By “infectious clone” or “infectious cDNA” of HPIV2 is meant cDNA or itsproduct, synthetic or otherwise, as well as RNA capable of beingdirectly incorporated into infectious virions which can be transcribedinto genomic or antigenomic HPIV2 RNA capable of serving as a templateto produce the genome of infectious HPIV2 viral or subviral particles.As noted above, defined mutations can be introduced into an infectiousHPIV2 clone by a variety of conventional techniques (e.g., site-directedmutagenesis) into a cDNA copy of the genome or antigenome. The use ofgenomic or antigenomic cDNA subfragments to assemble a complete genomeor antigenome cDNA as described herein has the advantage that eachregion can be manipulated separately, where small cDNA constructsprovide for better ease of manipulation than large cDNA constructs, andthen readily assembled into a complete cDNA.

Isolated polynucleotides (e.g., cDNA) encoding the HPIV2 genome orantigenome may be inserted into appropriate host cells by transfection,electroporation, mechanical insertion, transduction or the like, intocells which are capable of supporting a productive HPIV2 infection,e.g., HEp-2, FRhL-DBS2, LLC-MK2, MRC-5, and Vero cells. Transfection ofisolated polynucleotide sequences maybe introduced into cultured cellsby, for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro et al., Somatic Cell Genetics 7:603, 1981;Graham et al., Virology 52:456, 1973, electroporation (Neumann et al.,EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel etal., (ed.) Current Protocols in Molecular Biology, John Wiley and Sons,Inc., NY, 1987), cationic lipid-mediated transfection (Hawley-Nelson etal., Focus 15:73-79, 1993) or a commercially available transfectionregent, e.g., Lipofectamine-2000 (Invitrogen, Carlsbad, Calif.) or thelike (each of the foregoing references are incorporated herein byreference in its entirety).

By providing infectious clones of HPIV2, the invention permits a widerange of alterations to be recombinantly produced within the HPIV2genome (or antigenome), yielding defined mutations that specify desiredphenotypic changes. The compositions and methods of the invention forproducing recombinant HPIV2 permit ready detailed analysis andmanipulation of HPIV2 molecular biology and pathogenic mechanisms using,e.g., defined mutations to alter the function or expression of selectedHPIV2 proteins. Using these methods and compositions, one can readilydistinguish mutations responsible for desired phenotypic changes fromsilent incidental mutations, and select phenotype-specific mutations forincorporation into a recombinant HPIV2 genome or antigenome. In thiscontext, a variety of nucleotide insertions, deletions, substitutions,and rearrangements can be made in the HPIV2 genome or antigenome duringor after construction of the cDNA. For example, specific desirednucleotide sequences can be synthesized and, inserted at appropriateregions in the cDNA using convenient restriction enzyme sites.Alternatively, such techniques as site-specific mutagenesis, alaninescanning, PCR mutagenesis, or other such techniques well known in theart can be used to introduce mutations into the cDNA.

Recombinant modifications of HPIV2 provided within the invention aredirected toward the production of improved candidate viruses, e.g., toenhance viral attenuation and immunogenicity, to ablate epitopesassociated with undesirable immunopathology, to accommodate antigenicdrift, etc. To achieve these and other objectives, the compositions andmethods of the invention allow for a wide variety of modifications to beintroduced into a HPIV2 genome or antigenome for incorporation intoinfectious, recombinant HPIV2. For example, foreign genes or genesegments encoding antigenic determinants (e.g., protective antigens orimmunogenic epitopes) may be added within a HPIV2 clone to generaterecombinant HPIV2 viruses capable of inducing immunity to both HPIV2 andanother virus or pathogenic agent from which the antigenicdeterminant(s) was/were derived. Alternatively, foreign genes may beinserted, in whole or in part, encoding modulators of the immune system,such as cytokines, to enhance immunogenicity of a candidate virus. Othermutations that may be included within HPIV2 clones of the inventioninclude, for example, substitution of heterologous genes or genesegments (e.g., a gene segment encoding a cytoplasmic tail of aglycoprotein gene) with a counterpart gene or gene segment in a PIVclone. Alternatively, the relative order of genes within a HPIV2 clonecan be changed, a HPIV2 genome promoter or other regulatory element canbe replaced with its antigenome counterpart, or selected HPIV2 gene(s)rendered non-functional (e.g., by functional ablation involvingintroduction of a stop codon to prevent expression of the gene). Othermodifications in a HPIV2 clone can be made to facilitate manipulations,such as the insertion of unique restriction sites in various non-codingor coding regions of the HPIV2 genome or antigenome. In addition,nontranslated gene sequences can be removed to increase capacity forinserting foreign sequences.

As noted above, it is often desirable to adjust the phenotype ofrecombinant HPIV2 viruses for use by introducing additional mutationsthat increase or decrease attenuation or otherwise alter the phenotypeof the recombinant virus. Detailed descriptions of the materials andmethods for producing recombinant PIV from cDNA, and for making andtesting various mutations and nucleotide modifications set forth hereinas supplemental aspects of the present invention are provided in, e.g.,Durbin et al., Virology 235:323-332, 1997; U.S. patent application Ser.No. 09/083,793, filed May 22, 1998; U.S. patent application Ser. No.09/458,813, filed Dec. 10, 1999; U.S. patent application Ser. No.09/459,062, filed Dec. 10, 1999; U.S. Provisional Application No.60/047,575, filed May 23, 1997 (corresponding to InternationalPublication No. WO 98/53078), and U.S. Provisional Application No.60/059,385, filed Sep. 19, 1997, each incorporated herein by reference.

In particular, these incorporated references describe methods andprocedures for mutagenizing, isolating and characterizing PIV to obtainattenuated mutant strains (e.g., temperature sensitive (ts), coldpassaged (cp) cold-adapted (ca), small plaque (sp) and host-rangerestricted (hr) mutant strains) and for identifying the genetic changesthat specify the attenuated phenotype. In conjunction with thesemethods, the foregoing incorporated references detail procedures fordetermining replication, immunogenicity, genetic stability andimmunogenic efficacy of biologically derived and recombinantly producedattenuated HPIVs in accepted model systems reasonably correlative ofhuman activity, including hamster or rodent and non-human primate modelsystems. In addition, these references describe general methods fordeveloping and testing immunogenic compositions, including monovalentand bivalent immunogenic compositions against HPIV. Methods forproducing infectious recombinant HPIV3 by construction and expression ofcDNA encoding a PIV genome or antigenome coexpressed with essential PIVproteins are also described in the above-incorporated references, whichinclude description of the following exemplary plasmids that may beemployed to produce infectious HPIV3 clones: p3/7(131) (ATCC 97990);p3/7(131)2G (ATCC 97889); and p218(131) (ATCC 97991); each depositedunder the terms of the Budapest Treaty with the American Type CultureCollection (ATCC) of 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and granted the above identified accession numbers(deposits incorporated herein by reference). Methods for producinginfectious recombinant HPIV1 by construction and expression of cDNAencoding a HPIV1 recombinant or chimeric genome or antigenomecoexpressed with essential PIV proteins are similarly described in U.S.Provisional Application No. 60/331,961, filed Nov. 21, 2001, and inNewman et al., Virus Genes 24:77-92, 2002, each incorporated herein byreference.

Also disclosed in the above-incorporated references are methods forconstructing and evaluating infectious recombinant HPIV that aremodified to incorporate phenotype-specific mutations identified inbiologically derived PIV mutants, e.g., cold passaged (cp), cold adapted(ca), host range restricted (hr), small plaque (sp), and/or temperaturesensitive (ts) mutants, for example the HPIV3 JS cp45 mutant strain. TheHPIV3 JS cp45 strain has been deposited under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC) of 10801University Boulevard, Manassas, Va. 20110-2209, U.S.A. under PatentDeposit Designation PTA-2419 (deposit incorporated herein by reference).Mutations identified in this and other heterologous mutants viruses canbe readily incorporated into recombinant HPIV2 of the instant invention,as described herein below.

As exemplified in FIG. 1 (Panels A-E) various mutations identified in aheterologous negative stranded RNA virus can be incorporated intorecombinant HPIV2 candidates of the invention to yield attenuation orother desired phenotypic changes. The figure provides exemplary sequencealignments between HPIV2 wild-type (wt), HPIV3 wt, HPIV1 wt, or BPIV3 wtidentifying regions containing known attenuating mutations in theheterologous virus. Based on these and similar comparisons, mutationspreviously identified in a heterologous PIV or non-PIV virus are mappedto a corresponding position in HPIV2 for “transfer” (i.e, introductionof an identical, conservative or non-conservative mutation, potentiallyincluding a substitution, deletion or insertion, at a homologous orcorresponding position identified by the alignment) into recombinantHPIV2 of the invention. A large assemblage of such mutations areavailable (see, e.g., Newman et al., Virus Genes 24:77-92, 2002; Felleret al., Virology 276: 190-201, 2000; Skiadopoulos et al., Virology260:125-35, 1999; and Durbin et al., Virology 261:319-30, 1999, eachincorporated herein by reference) for incorporation into recombinantHPIV2 and chimeric HPIV2 candidates of the invention. As depicted inFIG. 1, a corresponding amino acid position previously shown to confer aphenotypic change in a heterologous virus (when the indicated wild-typeresidue is altered, e.g., by substitution) is identified by conventionalsequence alignment. The corresponding amino acid position in HPIV2 isthereby identified as a target site for mutation in a recombinant HPIV2to yield attenuation or other desired phenotypic changes.

In certain detailed embodiments, the HPIV2 genome or antigenome isrecombinantly modified to incorporate one or any combination ofmutation(s) selected from mutations specifying previously identifiedamino acid substitution(s) in the L protein at a position correspondingto Tyr942, Leu992, and/or Thr1558 of HPIV3 JS cp45. Correspondingtargets of wild-type (wt) HPIV2 L for incorporation of these exemplarymutations are Tyr948, Ala998, and Leu1566. In other embodiments, therecombinant HPIV2 genome or antigenome is modified to incorporate anattenuating mutation at an amino acid position corresponding to an aminoacid position of an attenuating mutation identified in a heterologous,mutant nonsegmented negative stranded RNA virus, for example, anotherHPIV, a non-human PIV such as a bovine PIV3 (BPIV3) or murine PIV11(MPIV1), or a non-PIV virus such as respiratory syncytial virus (RSV).In exemplary embodiments, an attenuating mutation identified in a BPIV3virus L protein, at amino acid position Thr1711, is incorporated in arecombinant HPIV2 of the invention at the corresponding position(Ser1724), as identified by conventional alignment methods (FIG. 1,Panels A-E).

In more specific embodiments, the HPIV2 genome or antigenomeincorporates one or any combination of mutation(s) selected frommutations specifying amino acid substitution(s) in the L protein ofTyr948His, Ala998Phe, Leu1566Ile, and/or Ser1724Ile of HPIV2 L, (FIG. 1,Panels B-E). Other mutations, including substitutions and deletions, atthe indicated target site for mutation, particularly those that areconservative to the foregoing exemplary mutations, are useful to achievedesired attenuation in recombinant HPIV2 candidates.

Considering, for example, mutations in non-human PIV viruses for usewithin the invention, the Kansas strain of BPIV3 was studied hereinbased on its known restriction in replication in the respiratory tractof humans and other primates. To identify the genetic determinants ofthe host-range attenuation phenotype of BPIV3 in primates, theantigenomic cDNA of HPIV3 was modified to contain the N, P, M, or L ORFof BPIV3 in place of the analogous HPIV3 ORF. In addition, the F and HNgenes were transferred together as a pair to replace their HPIV3counterparts, as described previously (Schmidt et al., J. Virol.74:8922-9, 2000, incorporated herein by reference). Each of the chimericbovine-human PIV3s containing the N, P, M, F and HN, or L was recoveredfrom cDNA. The recombinant chimeric viruses were biologically cloned byplaque isolation and vRNA was isolated from the cloned virus and wasused as a template to generate RT-PCR products. The structure of thegenome flanking the substituted ORF was confirmed for each recombinantvirus by sequencing and restriction enzyme analysis.

Two recombinant viruses, rHPIV3-L_(B T1711I) and rHPIV3-L_(B), bearingthe BPIV3 L ORF were generated. rHPIV3-L_(B T1711I) was generated first,but after it was found to be highly temperature sensitive in itsreplication in vitro, it was sequenced and was found to contain twopoint mutations in the L ORF that resulted in an Ala-425 to Val (A425V)and a Thr-1711 to Ile substitution (T1711I). The latter mutation waspresent in the antigenomic cDNA, but the former was a spontaneousmutation occurring following transfection of this cDNA. Anotherrecombinant was generated (rHPIV3 L_(B)) that had the authentic BPIV3 LORF sequence.

The kinetics of replication of each chimeric rHPIV3 in vitro wascompared to that of their wild type rHPIV3 and BPIV3 parent viruses byinfecting LLC-MK2 cells at an m.o.i. of 0.01 and measuring virus yieldat 24 hour intervals. Except for rHPIV3-L_(B T1711I) all of the chimericrHPIV3s bearing BPIV3 ORF substitutions grew at a rate similar to thatof their parent viruses, and all of the chimeric viruses grew to over10⁷ TCID₅₀/ml by day 5 post-infection. This confirmed that each of thesubstituted wild type BPIV3 proteins exhibited a high degree ofcompatibility with the proteins and cis-acting signals of the HPIV3backbone. In contrast, the restricted replication of rHPIV3-L_(B T1711I)in vitro indicates one or both of the amino acid substitutions in itsBPIV3 L polymerase protein are attenuating in vitro.

Considering these results, it was of interest to further characterizethe restricted replication exhibited by the mutant rHPIV3-L_(B T1711I) avirus. To determine if the ORF substitutions in each rHPIV3 altered theability of these viruses to grow at elevated temperatures, the level oftemperature sensitivity of replication of each chimeric rHPIV3 wascompared to that of the parent viruses and that of rHPIV3 cp45, which isa well characterized ts and attenuated candidate HPIV3 vaccine that waspreviously shown to be appropriately attenuated and immunogenic inhumans and non-human primates. The wild type and chimeric rHPIV3s wereevaluated for their ability to grow on LLC-MK2 cells at the permissivetemperature of 32° C. and at a range of higher temperatures.Surprisingly, both rHPIV3-L_(B) and rHPIV3-L_(B T1711I) were highly ts,with rHPIV3-L_(B T1711I) being more ts than either rHPIV3-L_(B) orrHPIV3 cp45. As noted above, the BPIV3 L ORF present in therHPIV3-L_(B T1711I) virus contained two amino acid coding changesrelative to wild type BPIV3 and it was of interest to determine whichone, or both, was responsible for the increased ts phenotype ofrHPIV3-L_(B T1711I) compared to rHPIV3-L_(B). An alternative viral cloneof rHPIV3-L_(B T1711I) was identified that contained the T1711Isubstitution but not the A425V mutation. This virus had the sameshut-off temperature as rHPIV3-L_(B T1711I), indicating that the T1711Imutation alone is responsible for the increased level of temperaturesensitivity of rHPIV3-L_(B T1711I.)

Each of the above-noted BPIV3 ORFs conferred restriction of replicationin the upper or lower respiratory tract of rhesus monkeys whensubstituted for the analogous ORF in HPIV3, demonstrating that thehost-range attenuation phenotype of BPIV3 is polygenic. Comparing themean peak titer of virus replication in the upper respiratory tract, thechimeric rHPIV3s fell into three groups: (i) viruses bearing the BPIV3 Mor L ORF or the F and HN genes were restricted approximately 16 to32-fold; (ii) viruses bearing the BPIV3 N or L_(T1711I) ORF exhibited a40-100 fold restriction of replication; and (iii) the chimeric HPIV3with the BPIV3 P ORF substitution exhibited a 1000-fold restriction inreplication, suggesting that the BPIV3 P ORF is the major contributor tothe attenuation phenotype. The level of replication of rHPIV3-P_(B) waseven lower than its BPIV3 parent virus in the upper respiratory tract,suggesting that some of its restricted replication in vivo may also bedue to a gene incompatibility effect that was not evident in vitro. Asimilar pattern of host range restriction for the panel of chimericviruses was observed in the lower respiratory tract. BecauserHPIV3-L_(B T1711I) was attenuated for replication in vitro and washighly ts, the level of attenuation of this virus observed in vivolikely is due to a combination of restricted replication specified byhost-range sequences and that specified by its high level of temperaturesensitivity (the body temperature of rhesus monkeys is about 39° C.).

To evaluate the immunogenicity of the rHPIV3s, serum samples werecollected prior to infection and on day 28 or 31 following infectionwith the chimeric rHPIV3s or their parent viruses, and the level ofserum HAI antibodies to HPIV3 was determined. Each of the chimericrecombinants bearing the HPIV3 F and HN glycoproteins induced a highlevel of HAI antibodies to HPIV3, whereas rHPIV3-F_(B)HN_(B) and theBPIV3 parent virus bearing the BPIV3 glycoproteins induced 8 to 16-foldless HAI antibody reactive with the human virus. rHPIV3 L_(B T1711I),rHPIV3-N_(B), and rHPIV3-P_(B) replicated approximately 2 to 10-foldless efficiently in the respiratory tract of rhesus monkeys compared toBPIV3, yet they induced approximately 4 to 32-fold more HPIV3 HAIantibodies, likely because they bear the homologous HPIV3 glycoproteins.

To evaluate the protective efficacy of the chimeric rHPIV3s, monkeyswere challenged IN and IT with 10⁶ TCID₅₀ of wild type HPIV3. NP and TLsamples were collected at 2-day intervals for 10 days post-challenge,and the virus present in the samples was quantified on LLC-MK2 cells.Each of the chimeric recombinants tested afforded a high level ofprotection against HPIV3 replication, including the highly attenuatedrHPIV3-P_(B) and rHPIV3 L_(B T1711I). Analysis of serum samplescollected following the challenge showed that each of the groupsdeveloped a similar, high titer of HAI antibodies to HPIV3, indicatingthat all the monkeys had indeed been infected.

The high level of attenuation of rHPIV3-L_(B T1711I) in rhesus monkeysreflects the additivity of the restriction of replication specified byhost-range sequences of its L gene and one or both of the T1711I andA425V point mutations. The T1711I mutation was shown to confer the tsphenotype in vitro and thus likely also is responsible for theattenuation phenotype in vivo, although a contribution by the A425Vmutation cannot be excluded at this time. The increased attenuation ofrHPIV3-L_(B T1711I) versus rHPIV3-L_(B) illustrates that ts and hostrange determinants of attenuation can be combined to fine-tune the levelof attenuation of a candidate HPIV2 virus of the invention.

In certain other detailed embodiments of the invention, the recombinantHPIV2 genome or antigenome incorporates a recombinant modification thatspecifies an attenuating mutation at an amino acid positioncorresponding to an amino acid position of an attenuating mutationidentified in a non-PIV, heterologous, mutant nonsegmented negativestranded RNA virus. In exemplary embodiments, the heterologous, mutantnonsegmented negative stranded RNA virus is respiratory syncytial virus(RSV). In one specific embodiment, the attenuating mutation identifiedin RSV comprises an amino acid substitution of phenylalanine at position521 of the RSV L protein, which aligns with a conserved position in theHPIV2, HPIV3, and HPIV1 L proteins. In HPIV2, the conserved target sitefor mutation corresponds to the Phe460 of the HPIV2 L protein (FIG. 1,Panel A). In one exemplary embodiment, Phe460 of the HPIV2 L protein issubstituted to a Leu residue or, alternatively, to another amino acid.

Many of the foregoing exemplary mutations which can be engineered in arecombinant HPIV2 candidate of the invention have been successfullyengineered and recovered in recombinant HPIV3 based candidates (Durbinet al., Virology 235:323-332, 1997; Skiadopoulos et al., J. Virol.72:1762-1768, 1998; Skiadopoulos et al., J. Virol. 73:1374-1381, 1999;U.S. patent application Ser. No. 09/083,793, filed May 22, 1998; U.S.patent application Ser. No. 09/458,813, filed Dec. 10, 1999; U.S. patentapplication Ser. No. 09/459,062, filed Dec. 10, 1999; U.S. ProvisionalApplication No. 60/047,575, filed May 23, 1997 (corresponding toInternational Publication No. WO 98/53078), and U.S. ProvisionalApplication No. 60/059,385, filed Sep. 19, 1997, each incorporatedherein by reference). In addition, the above-incorporated referencesdescribe construction of chimeric PIV recombinants, e.g., having the HNand F genes of HPIV1 substituted into a partial HPIV3 background genomeor antigenome, which is further modified to bear one or more of theattenuating mutations identified in HPIV3 JS cp45. One such chimericrecombinant incorporates all of the attenuating mutations identified inthe L gene of cp45. It has since been shown that all of the cp45mutations outside of the heterologous (HPIV1) HN and F genes can beincorporated in a HPIV3-1 recombinant to yield an attenuated, chimericcandidate.

Yet additional mutations that may be incorporated in recombinant HPIV2of the invention are mutations, e.g., attenuating mutations, identifiedin non-PIV pathogens, particularly other nonsegmented negative strandedRNA viruses besides PIV. In each of these contexts, attenuating andother desired mutations identified in one negative stranded RNA virusmay be “transferred”, e.g., copied, to a corresponding position withinthe genome or antigenome of a recombinant HPIV2 of the invention.Briefly, desired mutations in one heterologous negative stranded RNAvirus are transferred to the recombinant HPIV2 recipient (either in a“vector” HPIV2 genome or antigenome or in the heterologous “donor” geneor genome segment). This involves mapping the mutation in theheterologous mutant virus, identifying by routine sequence alignment thecorresponding site in the recipient, recombinant HPIV2, and mutating thenative sequence in the recombinant HPIV2, typically to correspond to anidentical or conservative mutation to the heterologous mutant genotype,as described in International Application No. PCT/US00/09695, filed Apr.12, 2000, published as WO 00/61737 on Oct. 19, 2000 corresponding toU.S. National Phase application Ser. No. 09/958,292, filed on Jan. 8,2002, and claiming priority to U.S. Provisional Patent Application Ser.No. 60/129,006, filed on Apr. 13, 1999, each incorporated herein byreference. Additional description pertaining to this aspect of theinvention is provided in Newman et al., Virus Genes 24:77-92, 2002;Feller et al., Virology 10; 276:190-201, 2000; Skiadopoulos et al.,Virology 260:125-35, 1999; and Durbin et al., Virology 261:319-30, 1999,each incorporated herein by reference.

It is often desired to modify the recipient recombinant HPIV2 genome orantigenome to encode an alteration at the subject site of mutation thatcorresponds conservatively to the alteration identified in theheterologous mutant virus. For example, if an amino acid substitutionmarks a site of mutation in the mutant virus compared to thecorresponding wild-type sequence, then a similar substitution can beengineered at the corresponding residue(s) in the recombinant HPIV2.Preferably the substitution will specify an identical or conservativeamino acid to the substitute residue present in the mutant viralprotein. However, it is also possible to alter the native amino acidresidue at the site of mutation non-conservatively with respect to thesubstitute residue in the mutant protein (e.g., by using any other aminoacid to disrupt or impair the function of the wild-type residue).Negative stranded RNA viruses from which exemplary mutations areidentified and transferred into a recombinant HPIV2 of the inventioninclude other PIVs (e.g., HPIV1, HPIV3, BPIV3 and MPIV1), RSV, Newcastledisease virus (NDV), simian virus 5 (SV5), measles virus (MeV),rinderpest virus, canine distemper virus (CDV), rabies virus (RaV) andvesicular stomatitis virus (VSV), among others.

Attenuating mutations in biologically derived PIV and other nonsegmentednegative stranded RNA viruses for incorporation within recombinant HPIV2of the invention may occur naturally or may be introduced into wild-typePIV strains and thereafter identified and characterized by well knownmutagenesis and analytic procedures. For example, incompletelyattenuated parental PIV or other heterologous viral mutant strains canbe produced by chemical mutagenesis during virus growth in cell culturesto which a chemical mutagen has been added, by selection of virus thathas been subjected to passage at suboptimal temperatures in order tointroduce growth restriction mutations, or by selection of a mutagenizedvirus that produces small plaques (sp) in cell culture, as described inthe above incorporated references.

By “biologically derived” is meant any virus not produced by recombinantmeans. Thus, biologically derived PIV include all naturally occurringPIV, including, e.g., naturally occurring PIV having a wild-type genomicsequence and PIV having allelic or mutant genomic variations from areference wild-type PIV sequence, e.g., PIV having a mutation specifyingan attenuated phenotype. Likewise, biologically derived PIV include PIVmutants derived from a parental PIV by, inter alia, artificialmutagenesis and selection procedures not involving direct recombinantDNA manipulation.

As noted above, production of a sufficiently attenuated biologicallyderived PIV or other viral mutant can be accomplished by several knownmethods. One such procedure involves subjecting a partially attenuatedvirus to passage in cell culture at progressively lower, attenuatingtemperatures. For example, partially attenuated mutants are produced bypassage in cell cultures at suboptimal temperatures. Thus, acold-adapted (ca) mutant or other partially attenuated PIV strain isadapted to efficient growth at a lower temperature by passage inculture. This selection of mutant PIV during cold-passage substantiallyreduces any residual virulence in the derivative strains as compared tothe partially attenuated parent. Alternatively, specific mutations canbe introduced into biologically derived PIV by subjecting a partiallyattenuated parent virus to chemical mutagenesis, e.g., to introduce tsmutations or, in the case of viruses which are already ts, additional tsmutations sufficient to confer increased attenuation and/or stability ofthe ts phenotype of the attenuated derivative. Means for theintroduction of ts mutations into PIV include replication of the virusin the presence of a mutagen such as 5-fluorouridine according togenerally known procedures. Other chemical mutagens can also be used.Attenuation can result from a ts mutation in almost any PIV gene,although a particularly amenable target for this purpose has been foundto be the polymerase (L) gene. The level of temperature sensitivity ofreplication in exemplary attenuated PIV for use within the invention isdetermined by comparing its replication at a permissive temperature withthat at several restrictive temperatures. The lowest temperature atwhich the replication of the virus is reduced 100-fold or more incomparison with its replication at the permissive temperature is termedthe shutoff temperature. In experimental animals and humans, both thereplication and virulence of PIV correlate with the mutant's shutofftemperature.

From biologically derived PIV and other nonsegmented negative strandedRNA viruses, a large “menu” of attenuating mutations is identifiable bythe teachings herein, each of which can be combined with any othermutation(s) for adjusting the level of attenuation, immunogenicity andgenetic stability in recombinant HPIV2 of the invention. In thiscontext, many recombinant HPIV2 candidates will include one or more, andpreferably two or more, mutations from a biologically derived PIV orother heterologous viral mutant, e.g., any one or combination ofmutations identified above from HPIV3 JS cp45, BPIV3, and RSV. Preferredrecombinant HPIV2 viruses within the invention will incorporate aplurality of mutations thus identified. Often, these mutations arestabilized against reversion in recombinant HPIV2 by multiple nucleotidesubstitutions in a codon specifying each mutation.

Mutations compiled into a “menu” as described above are introduced asdesired, singly or in combination, to adjust recombinant HPIV2 of theinvention to an appropriate level of attenuation, immunogenicity,genetic resistance to reversion from an attenuated phenotype, etc. Inaccordance with the foregoing description, the ability to produceinfectious recombinant HPIV2 from cDNA permits introduction of specificengineered changes within the recombinant HPIV2. In particular,infectious, recombinant HPIV2 viruses can be employed for furtheridentification of specific mutation(s) in biologically derived,attenuated HPIV2 strains, for example mutations that specify ts, ca, attand other phenotypes. Desired mutations identified by this and othermethods are introduced into recombinant HPIV2 candidate strains. Thecapability of producing virus from cDNA allows for routine incorporationof these mutations, individually or in various selected combinations,into a full-length cDNA clone, where after the phenotypes of rescuedrecombinant viruses containing the introduced mutations to be readilydetermined.

By identifying and incorporating specific mutations associated withdesired phenotypes, e.g., a ca or ts phenotype, into infectiousrecombinant HPIV2, the invention provides for other, site-specificmodifications at, or within close proximity to, the identified mutation.Whereas most attenuating mutations produced in biologically derived PIVsare single nucleotide changes, other “site specific” mutations can alsobe incorporated by recombinant techniques into a recombinant HPIV2. Asused herein, site-specific mutations include insertions, substitutions,deletions or rearrangements of from 1 to 3, up to about 5-15 or morealtered nucleotides (e.g., altered from a wild-type PIV sequence, from asequence of a selected mutant PIV strain, or from a parent recombinantPIV clone subjected to mutagenesis). Such site-specific mutations may beincorporated at, or within the region of, a selected, biologicallyderived point mutation. Alternatively, the mutations can be introducedin various other contexts within a recombinant HPIV2 clone, for exampleat or near a cis-acting regulatory sequence or nucleotide sequenceencoding a protein active site, binding site, immunogenic epitope, etc.

Site-specific recombinant HPIV2 mutants typically retain a desiredattenuating phenotype, but may additionally exhibit altered phenotypiccharacteristics unrelated to attenuation, e.g., enhanced or broadenedimmunogenicity, and/or improved growth. Further examples of desired,site-specific mutants include recombinant HPIV2 mutants engineered toincorporate additional, stabilizing nucleotide mutations in a codonspecifying an attenuating point mutation. Where possible, two or morenucleotide substitutions are introduced at codons that specifyattenuating amino acid changes in a parent mutant or recombinant HPIV2clone, yielding a recombinant HPIV2 with greater genetic resistance toreversion from an attenuated phenotype. In other embodiments,site-specific nucleotide substitutions, additions, deletions orrearrangements are introduced upstream or downstream, e.g., from 1 to 3,5-10 and up to 15 nucleotides or more 5′ or 3′, relative to a targetednucleotide position, e.g., to construct or ablate an existing cis-actingregulatory element.

In addition to single and multiple point mutations and site-specificmutations, changes to the recombinant HPIV2 disclosed herein includedeletions, insertions, substitutions or rearrangements of one or moregene(s) or genome segment(s). Particularly useful are deletionsinvolving one or more gene(s) or genome segment(s), which deletions havebeen shown to yield additional desired phenotypic effects (see, e.g.,U.S. patent application Ser. No. 09/350,821, filed by Durbin et al. onJul. 9, 1999, incorporated herein by reference). For example, in HPIV1,expression of one or more of the C, C′, Y1, and/or Y2 open readingframe(s) (ORF(s) or other auxiliary gene) can be reduced or ablated bymodifying the recombinant HPIV1 genome or antigenome, e.g., toincorporate a mutation that alters the coding assignment of aninitiation codon or mutation(s) that introduce one or one or more stopcodon(s). Alternatively, one or more of the C, C′, Y1, and/or Y2 ORF(s)or other auxiliary gene can be deleted in whole or in part to render thecorresponding protein(s) partially or entirely non-functional or todisrupt protein expression altogether. Recombinant HPIV2, which lack theC, C′, Y1, and/or Y2 ORF(s), can be modified in a similar fashion todelete or reduce expression of a gene, typically by deleting ormodifying an auxiliary gene or genome segment, such as the V ORF, andthese recombinants will possess highly desirable phenotypiccharacteristics for development of immunogenic compositions. Forexample, these modifications may specify one or more desired phenotypicchanges including (i) altered growth properties in cell culture, (ii)attenuation in the upper and/or lower respiratory tract of mammals,(iii) a change in viral plaque size, (iv) a change in cytopathic effect,and (v) a change in immunogenicity.

Thus, in more detailed aspects of the instant invention, a recombinantHPIV2 incorporates one or more partial or complete gene deletions, knockout mutations, or mutations that simply reduce or increase expression ofan HPIV2 gene. This can be achieved, e.g., by introducing a frame shiftmutation or termination codon within a selected coding sequence,altering translational start sites, changing the position of a gene orintroducing an upstream start codon to alter its rate of expression,changing GS and/or GE transcription signals to alter phenotype, ormodifying an RNA editing site (e.g., growth, temperature restrictions ontranscription, etc.). In more detailed aspects of the invention,recombinant HPIV2 viruses are provided in which expression of one ormore gene(s), e.g., a V ORF, is ablated at the translational ortranscriptional level without deletion of the gene or of a segmentthereof, by, e.g., introducing multiple translational termination codonsinto a translational open reading frame, altering an initiation codon,or modifying an editing site. These forms of knock-out virus will oftenexhibit reduced growth rates and small plaque sizes in tissue culture.Thus, these methods provide yet additional, novel types of attenuatingmutations which ablate expression of a viral gene that is not one of themajor viral protective antigens. In this context, knock-out virusphenotypes produced without deletion of a gene or genome segment can bealternatively produced by deletion mutagenesis, as described, toeffectively preclude correcting mutations that may restore synthesis ofa target protein. Other gene knock-outs can be made using alternatedesigns and methods that are well known in the art (as described, forexample, in (Kretschmer et al., Virology 216:309-316, 1996; Radecke etal., Virology 217:418-421, 1996; Kato et al., EMBO J. 16:578-587, 1987;and Schneider et al., Virology 277:314-322, 1996, each incorporatedherein by reference).

Nucleotide modifications that may be introduced into recombinant HPIV2constructs of the invention may alter small numbers of bases (e.g., from15-30 bases, up to 35-50 bases or more), large blocks of nucleotides(e.g., 50-100, 100-300, 300-500, 500-1,000 bases), or nearly complete orcomplete genes (e.g., 1,000-1,500 nucleotides, 1,500-2,500 nucleotides,2,500-5,000, nucleotides, 5,000-6,0000 nucleotides or more) in thevector genome or antigenome or heterologous, donor gene or genomesegment, depending upon the nature of the change (i.e, a small number ofbases may be changed to insert or ablate an immunogenic epitope orchange a small genome segment, whereas large block(s) of bases areinvolved when genes or large genome segments are added, substituted,deleted or rearranged.

In related aspects, the invention provides for supplementation ofmutations adopted into a recombinant HPIV2 clone from biologicallyderived PIV, e.g., ca and is mutations, with additional types ofmutations involving the same or different genes in a further modifiedrecombinant HPIV2. Each of the HPIV2 genes can be selectively altered interms of expression levels, or can be added, deleted, substituted orrearranged, in whole or in part, alone or in combination with otherdesired modifications, to yield a recombinant HPIV2 exhibiting novelcharacteristics. Thus, in addition to or in combination with attenuatingmutations adopted from biologically derived PIV and/or non-PIV mutants,the present invention also provides a range of additional methods forattenuating or otherwise modifying the phenotype of a recombinant HPIV2based on recombinant engineering of infectious PIV clones. A variety ofalterations can be produced in an isolated polynucleotide sequenceencoding a targeted gene or genome segment, including a donor orrecipient gene or genome segment in a recombinant HPIV2 genome orantigenome for incorporation into infectious clones. More specifically,to achieve desired structural and phenotypic changes in recombinantHPIV2, the invention allows for introduction of modifications whichdelete, substitute, introduce, or rearrange a selected nucleotide ornucleotide sequence from a parent genome or antigenome, as well asmutations which delete, substitute, introduce or rearrange whole gene(s)or genome segment(s), within a recombinant HPIV2.

Thus provided are modifications in recombinant HPIV2 of the inventionwhich simply alter or ablate expression of a selected gene, e.g., byintroducing a termination codon within a selected PIV coding sequence oraltering its translational start site or RNA editing site, changing theposition of a PIV gene relative to an operably linked promoter,introducing an upstream start codon to alter rates of expression,modifying (e.g., by changing position, altering an existing sequence, orsubstituting an existing sequence with a heterologous sequence) GSand/or GE transcription signals to alter phenotype (e.g., growth,temperature restrictions on transcription, etc.), and various otherdeletions, substitutions, additions and rearrangements that specifyquantitative or qualitative changes in viral replication, transcriptionof selected gene(s), or translation of selected protein(s). In thiscontext, any PIV gene or genome segment which is not essential forgrowth can be ablated or otherwise modified in a recombinant PIV toyield desired effects on virulence, pathogenesis, immunogenicity andother phenotypic characters. In addition to coding sequences, noncoding,leader, trailer and intergenic regions can be similarly deleted,substituted or modified and their phenotypic effects readily analyzed,e.g., by the use of minireplicons, and the recombinant HPIV2 describedherein.

In addition to these changes, the order of genes in a recombinant HPIV2construct can be changed, a PIV genome promoter replaced with itsantigenome counterpart or vice versa, portions of genes removed orsubstituted, and even entire genes deleted. Different or additionalmodifications in the sequence can be made to facilitate manipulations,such as the insertion of unique restriction sites in various intergenicregions or elsewhere. Nontranslated gene sequences can be removed toincrease capacity for inserting foreign sequences.

Other mutations for incorporation into recombinant HPIV2 constructs ofthe invention include mutations directed toward cis-acting signals,which can be readily identified, e.g., by mutational analysis of PIVminigenomes. For example, insertional and deletional analysis of theleader, trailer and/or flanking sequences identifies viral promoters andtranscription signals and provides a series of mutations associated withvarying degrees of reduction of RNA replication or transcription.Saturation mutagenesis (whereby each position in turn is modified toeach of the nucleotide alternatives) of these cis-acting signals alsocan be employed to identify many mutations that affect RNA replicationor transcription. Any of these mutations can be inserted into a chimericPIV antigenome or genome as described herein. Evaluation andmanipulation of trans-acting proteins and cis-acting RNA sequences usingthe complete antigenome cDNA is assisted by the use of PIV minigenomesas described in the above-incorporated references.

Additional mutations within recombinant HPIV2 viruses of the inventionmay also include replacement of the 3′ end of genome with itscounterpart from antigenome or vice versa, which is associated withchanges in RNA replication and transcription. In one exemplaryembodiment, the level of expression of specific PIV proteins, such asthe protective HN and/or F antigens, can be increased by substitutingthe natural sequences with ones which have been made synthetically anddesigned to be consistent with efficient translation. In this context,it has been shown that codon usage can be a major factor in the level oftranslation of mammalian viral proteins (Haas et al., Current Biol.6:315-324, 1996, incorporated herein by reference). Optimization byrecombinant methods of the codon usage of the mRNAs encoding the HN andF proteins of recombinant HPIV2 will provide improved expression forthese genes.

In another exemplary embodiment, a sequence surrounding a translationalstart site (preferably including a nucleotide in the −3 positionrelative to the AUG start site) of a selected HPIV2 gene or donor geneincorporated in an HPIV2 vector is modified, alone or in combinationwith introduction of an upstream start codon, to modulate geneexpression by specifying up- or down-regulation of translation.Alternatively, or in combination with other recombinant modificationsdisclosed herein, gene expression of a recombinant HPIV2 can bemodulated by altering a transcriptional GS or GE signal of any selectedgene(s) of the virus. In alternative embodiments, levels of geneexpression in a recombinant HPIV2 candidate are modified at the level oftranscription. In one aspect, the position of a selected gene in the PIVgene map can be changed to a more promoter-proximal or promotor-distalposition, whereby the gene will be expressed more or less efficiently,respectively. According to this aspect, modulation of expression forspecific genes can be achieved yielding reductions or increases of geneexpression from two-fold, more typically four-fold, up to ten-fold ormore compared to wild-type levels often attended by a commensuratedecrease in expression levels for reciprocally, positionally substitutedgenes. These and other transpositioning changes yield novel recombinantHPIV2 viruses having attenuated phenotypes, for example due to decreasedexpression of selected viral proteins involved in RNA replication, orhaving other desirable properties such as increased antigen expression.

In other embodiments, recombinant HPIV2 viruses useful in immunogeniccompositions can be conveniently modified to accommodate antigenic driftin circulating virus. Typically the modification will be in the HNand/or F proteins. An entire HN or F gene, or a genome segment encodinga particular immunogenic region thereof, from one PIV (HPIV2 or anotherHPIV) strain or group is incorporated into a recombinant HPIV2 genome orantigenome cDNA by replacement of a corresponding region in a recipientclone of a different PIV strain or group, or by adding one or morecopies of the gene, such that multiple antigenic forms are represented.Progeny virus produced from the modified recombinant HPIV2 can then beused in immunization protocols against emerging PIV strains.

In certain aspects of the invention, replacement of a HPIV2 codingsequence or non-coding sequence (e.g., a promoter, gene-end, gene-start,intergenic or other cis-acting element) with a heterologous (e.g.,non-HPIV2) counterpart yields chimeric HPIV2 having a variety ofpossible attenuating and other phenotypic effects. For example, hostrange and other desired effects can be engineered by importing a bovinePIV3 (BPIV3) or murine PIV1 (MPIV1) protein, SV5, SV41, NDV, proteindomain, gene or genome segment into a recombinant HPIV2 “background”genome or antigenome, wherein the bovine or murine gene does notfunction efficiently in a human cell, e.g., from incompatibility of theheterologous sequence or protein with a biologically interactive HPIVsequence or protein (i.e, a sequence or protein that ordinarilycooperates with the substituted sequence or protein for viraltranscription, translation, assembly, etc.) or, more typically in a hostrange restriction, with a cellular protein or some other aspect of thecellular milieu which is different between the permissive and lesspermissive host. In exemplary embodiments, bovine PIV sequences areselected for introduction into HPIV2 based on known aspects of bovineand heterologous human PIV structure and function.

In more detailed aspects, the invention provides methods for attenuatingrecombinant HPIV2 candidates based on the construction of chimerasbetween HPIV2 and a non-human PIV, for example MPIV1 (Sendai virus),BPIV3, SV5, SV41, and NDV (e.g., as disclosed in U.S. patent applicationSer. No. 09/586,479, filed Jun. 1, 2000 by Schmidt et al. (correspondingto PCT Publication WO 01/04320); Schmidt et al., J. Virol. 74:8922-9,2000, each incorporated herein by reference). This method of attenuationis based on host range effects due to the introduction of one or moregene(s) or genome segment(s) of the non-human PIV into a human PIVvector-based chimeric virus. For example, there are numerous nucleotideand amino acid sequence differences between BPIV and HPIVs, which arereflected in host range differences. Between HPIV3 and BPIV3 the percentamino acid identity for each of the following proteins is: N (86%), P(65%), M (93%), F (83%), HN (77%), and L (91%). The host rangedifference is exemplified by the highly permissive growth of HPIV3 inrhesus monkeys, compared to the restricted replication of two differentstrains of BPIV3 in the same animal (van Wyke Coelingh et al., J.Infect. Dis. 157:655-662, 1988, incorporated herein by reference).Although the basis of the host range differences between HPIV3 and BPIV3remains to be fully elucidated, it has been shown to involve multiplegene and multiple amino acid differences. The involvement of multiplegenes and possibly cis-acting regulatory sequences, each involvingmultiple amino acid or nucleotide differences, gives a broad basis forattenuation, one which is highly stable to reversion. This is incontrast to the situation with other live attenuated HPIV3 viruses thatare attenuated by one or several point mutations. In this case,reversion of any individual mutation may yield a significantreacquisition of virulence or, in a case where only a single residuespecified attenuation, complete reacquisition of virulence. In exemplaryembodiments of the invention, the recombinant HPIV2 genome or antigenomeis combined with a heterologous gene or genome segment, such as an N, P,M, or L, ORF derived from a BPIV3, or another animal paramyxoviruses.

The above-incorporated references disclose that HPIV3/BPIV3 chimericrecombinants involving both bovine PIV3 strains Kansas (Ka) and shippingfever (SF) are viable and replicate as efficiently in cell culture aseither HPIV3 or BPIV3 parent-indicating that the chimeric recombinantsdid not exhibit gene incompatibilities that restricted replication invitro. This property of efficient replication in vitro is importantsince it permits efficient manufacture of this biological. Also, the Kaand the SF HPIV31BPIV3 chimeric recombinants (termed cKa and cSF),bearing only one bovine gene, are nearly equivalent to their BPIV3parents in the degree of host range restriction in the respiratory tractof the rhesus monkey. In particular, the cKa and cSF viruses exhibitapproximately a 60-fold or 30-fold reduction, respectively, inreplication in the upper respiratory tract of rhesus monkeys compared toreplication of HPIV3. Based on this finding, it is expected that otherBPIV3 genes will also confer desired levels of host range restrictionwithin chimeric HPIV2 candidates of the invention. Thus, according tothe methods herein, a list of attenuating determinants will be readilyidentified in heterologous genes and genome segments of BPIV3 and othernon-human PIVs that will confer, in appropriate combination, a desiredlevel of host range restriction and immunogenicity on recombinant HPIV2viruses.

Chimeric human-bovine or human-murine recombinant HPIV2 are thereforeprovided herein that include a partial or complete “background” HPIV2genome or antigenome derived from or patterned after HPIV2 combined withone or more heterologous gene(s) or genome segment(s) of a non-human PIVto form the chimeric PIV genome or antigenome. In preferred aspects ofthe invention, chimeric HPIV2 of this type incorporate a partial orcomplete HPIV2 background genome or antigenome combined with one or moreheterologous gene(s) or genome segment(s), e.g., from a bovine PIV. Thepartial or complete background genome or antigenome typically acts as arecipient backbone into which the heterologous genes or genome segmentsof the counterpart, non-human PIV are incorporated. Heterologous genesor genome segments from the counterpart PIV represent “donor” genes orpolynucleotides that are combined with, or substituted within, thebackground genome or antigenome to yield a chimeric HPIV2 that exhibitsnovel phenotypic characteristics compared to one or both of thecontributing PIVs. For example, addition or substitution of heterologousgenes or genome segments within a selected recipient HPIV2 strain mayresult in an increase or decrease in attenuation, growth changes,altered immunogenicity, or other desired phenotypic changes as comparedwith a corresponding phenotype(s) of the unmodified recipient and/ordonor (U.S. patent application Ser. No. 09/586,479, filed Jun. 1, 2000by Schmidt et al.; Schmidt et al., J. Virol. 74:8922-9, 2000, eachincorporated herein by reference).

Genes and genome segments that may be selected for use as heterologoussubstitutions or additions within chimeric PIV vectors include genes orgenome segments encoding a PIV N, V, P, M, F, HN and/or L protein(s) orportion(s) thereof. In addition, genes and genome segments encodingproteins found in other PIV viruses, as well as nonPIV proteins (e.g.,an SH protein as found in mumps, RSV, and SV5 viruses), may beincorporated within additional chimeric HPIV2 recombinants of theinvention. Regulatory regions, such as the extragenic 3′ leader or 5′trailer regions, and gene-start, gene-end, intergenic regions, or 3′ or5′ non-coding regions, are also useful as heterologous substitutions oradditions. In exemplary aspects, chimeric HPIV2 bearing one or morebovine or murine PIV gene(s) or genome segment(s) exhibit a high degreeof host range restriction, e.g., in the respiratory tract of mammalianmodels of human PIV infection such as hamsters and non-human primates.In more detailed embodiments HPIV2 is attenuated by the addition orsubstitution of one or more bovine PIV3 gene(s) or genome segment(s)selected from N, M, L, V, and P genes and genome segments to a partialor complete HPIV2 background genome or antigenome.

Preferably, the degree of host range restriction exhibited byhuman-bovine and other chimeric HPIV2 for use as candidates of theinvention is comparable to the degree of host range restrictionexhibited by the respective non-human PIV or other “donor” strain.Preferably, the restriction should have a true host range phenotype,i.e, it should be specific to the host in question and should notrestrict replication in vitro in a suitable cell line. In addition,chimeric HPIV2 bearing one or more bovine or murine PIV gene(s) orgenome segment(s) elicit a desired immunogenic response in hostssusceptible to HPIV2 infection. Thus, the invention provides a new basisfor attenuating a live HPIV2 virus vector for developing immunogeniccompositions against HPIV2 and other pathogens based on host rangeeffects.

In combination with the host range phenotypic effects provided in thehuman-non-human chimeric HPIV2 of the invention, it is often desirableto adjust the attenuation phenotype by introducing additional mutationsthat increase or decrease attenuation of the chimeric virus. Thus, inadditional aspects of the invention, attenuated, human-bovine orhuman-murine chimeric HPIV2 are produced in which the chimeric genome orantigenome is further modified by introducing one or more attenuatingmutations specifying an attenuating phenotype in the resultant virus orsubviral particle. These can include mutations generated de novo andtested for attenuating effects according to a rational designmutagenesis strategy. Alternatively, the attenuating mutations may beidentified in existing biologically derived mutant PIV and non-PIVviruses and thereafter incorporated into a human-bovine or human-murinechimeric HPIV2 of the invention. Exemplary mutations specify lesions inRNA regulatory sequences or in encoded proteins.

In preferred chimeric HPIV2 candidates of the invention, attenuationmarked by replication in the lower and/or upper respiratory tract in anaccepted animal model that is reasonably correlated with PIV replicationand immunogenic activity in humans (e.g., hamsters, rhesus monkeys orchimpanzees), is reduced by at least about two-fold, more often about5-fold, 10-fold, or 20-fold, and preferably 50-100-fold and up to1,000-fold or greater overall (e.g., as measured between 3-8 daysfollowing infection) compared to growth of the corresponding wild-typeor mutant parental PIV strain.

Within the methods of the invention, additional genes or genome segmentsmay be inserted into or proximate to a recombinant or chimeric HPIV2genome or antigenome. For example, various supernumerary heterologousgene(s) or genome segment(s) can be inserted at any of a variety ofsites within the recombinant genome or antigenome, for example at aposition 3′ to N, between the N/P, P/M, and/or HN/L genes, or at anotherintergenic junction or non-coding region of the HPIV2 vector genome orantigenome. Exemplary gene insertion details are illustrated in FIG. 2.The inserted genes may be under common control with recipient genes, ormay be under the control of an independent set of transcription signals.Genes of interest in this context include genes encoding cytokines, forexample, an interleukin (IL-2 through IL-18, e.g., interleukin 2 (IL-2),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL6),interleukin 18 (IL-18), tumor necrosis factor alpha (TNFa), interferongamma (IFNy), or granulocyte-macrophage colony stimulating factor(GM-CSF), as well as (see, e.g., U.S. application Ser. No. 09/614,285,filed Jul. 12, 2000 and priority U.S. Provisional Application Ser. No.60/143,425 filed Jul. 13, 1999, each incorporated herein by reference).Coexpression of these additional proteins provides the ability to modifyand improve immune responses against recombinant HPIV2 of the inventionquantitatively and/or qualitatively.

In other aspects of the invention, insertion of heterologous nucleotidesequences into recombinant HPIV2 candidates are employed separately tomodulate the level of attenuation of candidate recombinants, e.g., forthe upper respiratory tract. Thus, it is possible to insert nucleotidesequences into a recombinant HPIV2 that both direct the expression of aforeign protein and that attenuate the virus in an animal host, or touse nucleotide insertions separately to attenuate candidate viruses. Todefine some of the rules that govern the effect of gene insertion onattenuation, gene units of varying lengths may be inserted into a wildtype HPIV2 backbone and the effects of gene unit length on attenuationexamined. Novel gene unit insertions are contemplated in this regardthat do not contain a significant ORF, permitting identification of theeffect of gene unit length independently of an effect of the expressedprotein of that gene. These heterologous sequences may be inserted as anextra gene unit of various sizes, e.g., from about 150 or more nts inlength up to 3,000 nts or more in length. As demonstrated herein, geneinsertions or extensions larger than about 3,000 nts in length.

Gene unit (GU) insertions of about 1,000 or 2,000 nts in length willsubstantially attenuate rHPIV2 candidates for the upper respiratorytract of mammalian subjects. In addition, gene unit insertions can havethe dual effect of both attenuating a candidate virus and inducing animmunogenic response against a second virus. Alternately, geneextensions in the 3′-noncoding region (NCR) of a HPIV2 gene, whichcannot express additional proteins, can also be attenuating in and ofthemselves. Within these methods of the invention, gene insertion lengthis a determinant of attenuation.

GU and NCR insertions within recombinant HPIV2 of the invention producean attenuation phenotype characterized by efficient replication in vitroand decreased replication in vivo, a phenotype not previously describedfor other paramyxovirus insertions. The mechanism of attenuationresulting from a GU insertion may result from one or more of thefollowing factors acting predominantly in vivo. The addition of an extragene unit may decrease the level of transcription of downstream genessince there is a transcriptional gradient in which morepromoter-proximal genes are transcribed at a higher rate than the morepromoter-distal genes. The decreased expression of the downstream geneproducts resulting from the decreased abundance of their mRNAs couldresult in attenuation if their gene product is limiting or if a specificratio of gene products that is required for efficient replication isaltered. It is thought that the transcription gradient is a consequenceof the transcriptase complex falling off the template duringtranscription as well as during the transfer across gene junctions.Alternatively, the increase in the overall length of the genome and theextra mRNAs transcribed may increase the level of viral double strandedRNA made which in turn may induce a higher level of the antiviralactivity of the interferon system. Finally, the overall level of genomereplication may be reduced due to the increase in length of the genomeand the antigenome. This may result from a disengagement of replicasecomplexes from the template during replication of the genomic RNA orantigenomic RNA. The decreased amount of genome available for packaginginto virions may result in a decrease in virus yield that results inattenuation.

The mechanism of attenuation resulting from a NCR insertion may resultfrom one or more of the following factors. The extra length of the3′-end of HN mRNA resulting from the NCR insertion may contribute to theinstability of the mRNA and lead to a decrease in the expression of theHN protein. Alternatively, the increase in the overall length of thegenome and the extra length of the HN mRNA may increase the level ofviral double stranded RNA made that can induce a higher level of theantiviral activity of the interferon system. Alternatively oradditionally, the overall level of genome replication may be reduced dueto the increase in length of the genome and the antigenome. This mayresult from a disengagement of replicase complexes from the templateduring replication of the genomic RNA or antigenomic RNA. The decreasedamount of genome available for packaging into virions could result in adecrease in virus yield that results in attenuation. Finally, theaddition of extra nucleotides to the 3′ end of the HN gene coulddecrease the level of transcription of downstream genes since thetranscriptase complex could fall off the template during transcriptionof the extra nucleotides at the 3′ end of the HN gene.

Deletions, insertions, substitutions and other mutations involvingchanges of whole viral genes or genome segments within rHPIV2 of theinvention yield highly stable candidates, which are particularlyimportant in the case of immunosuppressed individuals. Many of thesechanges will result in attenuation of resultant strains, whereas otherswill specify different types of desired phenotypic changes. For example,accessory (i.e, not essential for in vitro growth) genes are excellentcandidates to encode proteins that specifically interfere with hostimmunity (see, e.g., Kato et al., EMBO. J. 16:578-87, 1997, incorporatedherein by reference). Ablation of such genes in candidate viruses isexpected to reduce virulence and pathogenesis and/or improveimmunogenicity.

In more detailed embodiments of the invention, chimeric HPIV2 virusesare constructed using a HPIV2 “vector” genome or antigenome that isrecombinantly modified to incorporate one or more antigenic determinantsof a heterologous pathogen. The vector genome or antigenome is comprisedof a partial or complete HPIV2 genome or antigenome, which may itselfincorporate nucleotide modifications such as attenuating mutations. Thevector genome or antigenome is modified to form a chimeric structurethrough incorporation of a heterologous gene or genome segment. Morespecifically, chimeric HPIV2 viruses of the invention are constructedthrough a cDNA-based virus recovery system that yields recombinantviruses that incorporate a partial or complete vector or “background”HPIV2 genome or antigenome combined with one or more “donor” nucleotidesequences encoding the heterologous antigenic determinant(s). Inexemplary embodiments a HPIV2 vector genome or antigenome is modified toincorporate one or more genes or genome segments that encode antigenicdeterminant(s) of one or more heterologous PIVs (e.g., HPIV 1 and/orHPIV3), and/or a non-PIV pathogen (e.g., RSV, human metapneumovirus, ormeasles virus). Thus constructed, chimeric HPIV2 viruses of theinvention may elicit an immune response against a specific PIV, e.g.,HPIV1, HPIV2, and/or HPIV3, or against a non-PIV pathogen.Alternatively, compositions and methods are provided employing aHPIV2-based chimeric virus to elicit a polyspecific immune responseagainst multiple PIVs, e.g., HPIV2 and HPIV3, or against one or moreHPIVs and a non-PIV pathogen such as measles virus. Exemplaryconstruction of a chimeric, vector HPIV2 candidate virus is illustratedin FIG. 2.

In preferred aspects of the invention, a chimeric HPIV2 in this contextincorporates a partial or complete human HPIV2 incorporating one or moreheterologous polynucleotide(s) encoding one or more antigenicdeterminants of the heterologous pathogen, which polynucleotides may beadded to or substituted within the HPIV2 vector genome or antigenome toyield the chimeric HPIV2 recombinant. The chimeric HPIV2 virus thusacquires the ability to elicit an immune response in a selected hostagainst the heterologous pathogen. In addition, the chimeric virus mayexhibit other novel phenotypic characteristics compared to one or bothof the vector PIV and heterologous pathogens.

The partial or complete vector genome or antigenome generally acts as abackbone into which heterologous genes or genome segments of a differentpathogen are incorporated. Often, the heterologous pathogen is adifferent PIV from which one or more gene(s) or genome segment(s) is/arecombined with, or substituted within, the vector genome or antigenome.In addition to providing novel immunogenic characteristics, the additionor substitution of heterologous genes or genome segments within thevector HPIV2 strain may confer an increase or decrease in attenuation,growth changes, or other desired phenotypic changes as compared with thecorresponding phenotype(s) of the unmodified vector and donor viruses.

Heterologous genes or genome segments of one PIV or non-PIV virus may beadded as a supernumerary genomic element to a partial or complete genomeor antigenome of HPIV2. Alternatively, one or more heterologous gene(s)or genome segment(s) of one PIV may be substituted at a positioncorresponding to a wild-type gene order position of a counterpartgene(s) or genome segment(s) that is deleted within the HPIV2 vectorgenome or antigenome. In yet additional embodiments, the heterologousgene or genome segment is added or substituted at a position that ismore promoter-proximal or promotor-distal compared to a wild-type geneorder position of the counterpart gene or genome segment within thevector genome or antigenome to enhance or reduce, respectively,expression of the heterologous gene or genome segment.

The introduction of heterologous immunogenic proteins, protein domainsand immunogenic epitopes to produce chimeric HPIV2 is particularlyuseful to generate novel immune responses in an immunized host. Additionor substitution of an immunogenic gene or genome segment from one, donorpathogen within a recipient HPIV2 vector genome or antigenome cangenerate an immune response directed against the donor pathogen, theHPIV2 vector, or against both the donor pathogen and vector.

General methods and compositions useful for engineering chimeric PIVviruses have been developed for HPIV3 (Durbin et al., Virology235:323-332, 1997; Skiadopoulos et al., J. Virol. 72:1762-1768, 1998;Tao et al., J Virol 72:2955-2961, 1998; Skiadopoulos et al., J. Virol.73:1374-1381, 1999; Skiadopoulos et al., Vaccine 18:503-510, 1999; Taoet al., Vaccine 17:1100-1108, 1999; Tao et al., Vaccine 18:1359-1366,2000; U.S. patent application Ser. No. 09/083,793, filed May 22, 1998;U.S. patent application Ser. No. 09/458,813, filed Dec. 10, 1999; U.S.patent application Ser. No. 09/459,062, filed Dec. 10, 1999; U.S.Provisional Application No. 60/047,575, filed May 23, 1997(corresponding to International Publication No. WO 98/53078), and U.S.Provisional Application No. 60/059,385, filed Sep. 19, 1997, eachincorporated herein by reference). In particular, the above-incorporatedreferences describe construction of chimeric PIV recombinants, e.g.,having the HN and F genes of HPIV1 substituted into a partial HPIV3background genome or antigenome, which is further modified to bear oneor more of the attenuating mutations identified in HPIV3 JS cp45. Onesuch chimeric recombinant incorporates all of the attenuating mutationsidentified in the L gene of cp45 outside of the heterologous (HPIV1) HNand F genes, yielding an attenuated, chimeric candidate.

However, it has been reported that prior infection with HPIV3 partiallyrestricts both the immunogenicity of HPIV3-1 recombinant viruses and theefficacy of such viruses against subsequent HPIV1 challenge. Thisrestriction appears to be due to an immune response against the HPIV3internal proteins that are shared by the two viruses (Tao et al.,Vaccine 17:1100-1108, 1999; Tao et al., Vaccine 18:1359-1366, 2000, eachincorporated herein by reference). The immune response against theinternal HPIV3 proteins was short-lived and did not appear to contributeto long-term efficacy, but it might be sufficient to interfere withsequential immunizations spaced at relatively short intervals such astwo months, as is envisioned for the live-attenuated RSV and PIVimmunogenic compositions (see Introduction). The HPIV2 reverse geneticssystem described here resolves this problem by providing live-attenuatedHPIV2 that will be infectious and immunogenic in infants that have beenpreviously exposed to HPIV3, as well as other viruses such as RSV.

Chimeric HPIV2 of the invention may also be constructed that express achimeric protein, for example an immunogenic glycoprotein having acytoplasmic tail and/or transmembrane domain specific to a HPIV2 vectorfused to a heterologous ectodomain of a different PIV or non-PIVpathogen to provide a fusion protein that elicits an immune responseagainst the heterologous pathogen. For example, a heterologous genomesegment encoding a glycoprotein ectodomain from a HPIV1 or HPIV3 HN or Fglycoprotein may be joined with a genome segment encoding thecorresponding HPIV2 HN or F glycoprotein cytoplasmic and transmembranedomains to form a HPIV2-1 or HPIV2-3 chimeric glycoprotein that elicitsan immune response against HPIV1 or HPIV3.

Briefly, HPIV2 of the invention expressing a chimeric glycoproteincomprise a major nucleocapsid (N) protein, a nucleocapsid phosphoprotein(P), a large polymerase protein (L), and a HPIV2 vector genome orantigenome that is modified to encode a chimeric glycoprotein. Thechimeric glycoprotein incorporates one or more heterologous antigenicdomains, fragments, or epitopes of a second, antigenically distinctHPIV. Preferably, this is achieved by substitution within the HPIV2vector genome or antigenome of one or more heterologous genome segmentsof the second HPIV that encode one or more antigenic domains, fragments,or epitopes, whereby the genome or antigenome encodes the chimericglycoprotein that is antigenically distinct from the parent, vectorvirus.

In more detailed aspects, the heterologous genome segment or segmentspreferably encode a glycoprotein ectodomain or immunogenic portion orepitope thereof, and optionally include other portions of theheterologous or “donor” glycoprotein, for example both an ectodomain andtransmembrane region that are substituted for counterpart glycoproteinecto-and transmembrane domains in the vector genome or antigenome.Preferred chimeric glycoproteins in this context may be selected fromHPIV HN and/or F glycoproteins, and the vector genome or antigenome maybe modified to encode multiple chimeric glycoproteins. In preferredembodiments, the HPIV2 vector genome or antigenome is a partial genomeor antigenome and the second, antigenically distinct HPIV is eitherHPIV1 or HPIV3. In one exemplary embodiment, both glycoproteinectodomain(s) of HPIV1 or HPIV3 HN and F glycoproteins are substitutedfor corresponding HN and F glycoprotein ectodomains in the HPIV2 vectorgenome or antigenome. In another exemplary embodiment, HPIV1 or HPIV3ectodomain and transmembrane regions of one or both FIN and/or Fglycoproteins are fused to one or more corresponding HPIV2 cytoplasmictail region(s) to form the chimeric glycoprotein. Further detailsconcerning these aspects of the invention are provided in U.S. patentapplication Ser. No. 09/459,062, filed on Dec. 10, 1999 by Tao et al.,incorporated herein by reference.

As used herein, the term “gene” generally refers to a portion of asubject genome, e.g., a HPIV2 genome, encoding an mRNA and typicallybegins at the upstream end with a gene-start (GS) signal and ends at thedownstream end with the gene-end (GE) signal. The term gene is alsointerchangeable with the term “translational open reading frame”, orORF, particularly in the case where a protein, such as the C protein, isexpressed from an additional ORF rather than from a unique mRNA. Theviral genome of all PIVs also contains extragenic leader and trailerregions, possessing part of the promoters required for viral replicationand transcription, as well as non-coding and intergenic regions.Transcription initiates at the 3′ end and proceeds by a sequentialstop-start mechanism that is guided by short conserved motifs found atthe gene boundaries. The upstream end of each gene contains a gene-start(GS) signal that directs initiation of its respective mRNA. Thedownstream terminus of each gene contains a gene-end (GE) motif thatdirects polyadenylation and termination.

To construct chimeric HPIV2 viruses of the invention, one or more PIVgene(s) or genome segment(s) may be deleted, inserted or substituted inwhole or in part. This means that partial or complete deletions,insertions and substitutions may include open reading frames and/orcis-acting regulatory sequences of any one or more of the PIV genes orgenome segments. By “genome segment” is meant any length of continuousnucleotides from the PIV genome, which might be part of an ORF, a gene,or an extragenic region, or a combination thereof. When a subject genomesegment encodes an antigenic determinant, the genome segment encodes atleast one immunogenic epitope capable of eliciting a humoral or cellmediated immune response in a mammalian host. The genome segment mayalso encode an immunogenic fragment or protein domain. In other aspects,the donor genome segment may encode multiple immunogenic domains orepitopes, including recombinantly synthesized sequences that comprisemultiple, repeating or different, immunogenic domains or epitopes.

Exemplary genome sequences of heterologous viruses for use within theseaspects of the invention have been described for the human PIV3 strainsJS (GenBank accession number Z11575, incorporated herein by reference)and Washington (Galinski M. S. In Kingsbury, D. W. (Ed.), TheParamyxoviruses, pp. 537-568, Plenum Press, New York, 1991, incorporatedherein by reference); for HPIV1/Wash64 (GenBank accession numberAF457102, incorporated herein by reference); for the bovine PIV3 (BPIV3)strain 910N (GenBank accession number D80487, incorporated herein byreference); for BPIV3 Kansas (GenBank accession number AF178654,incorporated herein by reference); for BPIV Shipping fever (GenBankaccession number AF178655, incorporated herein by reference); for RSV A2(GenBank accession number AF035006, incorporated herein by reference);and for hMPV (GenBank accession number AF371337, incorporated herein byreference).

In preferred embodiments of the invention, the chimeric HPIV2 bears oneor more major antigenic determinants of a human PIV, or multiple humanPIVs, including HPIV1, HPIV2 and/or HPIV3. These preferred candidateselicit an effective immune response in humans against one or moreselected HPIVs. As noted above, the antigenic determinant(s) thatelicit(s) an immune response against HPIV may be encoded by the HPIV2vector genome or antigenome, or may be inserted within or joined to thePIV vector genome or antigenome as a heterologous gene or gene segment.The major protective antigens of human PIVs are their HN and Fglycoproteins. However, all PIV genes are candidates for encodingantigenic determinants of interest, including internal protein genesthat may encode such determinants as, for example, CTL epitopes.

Preferred chimeric HPIV2 candidate viruses of the invention bear one ormore major antigenic determinants from each of a plurality of HPIVs orfrom a HPIV and a non-PIV pathogen. Chimeric HPIV2 viruses thusconstructed include one or more heterologous gene(s) or genomesegment(s) encoding antigenic determinant(s) of the same or aheterologous (for example HPIV1 or HPIV3) PIV. These and otherconstructs yield chimeric PIVs that elicit either a mono- orpoly-specific immune response in humans to one or more HPIVs. Furtherdetailed aspects of the invention are provided in U.S. patentapplication Ser. No. 09/083,793, filed May 22, 1998; U.S. patentapplication Ser. No. 09/458,813, filed Dec. 10, 1999; U.S. patentapplication Ser. No. 09/459,062, filed Dec. 10, 1999; U.S. ProvisionalApplication No. 60/047,575, filed May 23, 1997 (corresponding toInternational Publication No. WO 98/53078), U.S. Provisional ApplicationNo. 60/059,385, filed Sep. 19, 1997; U.S. Provisional Application No.60/170,195 filed Dec. 10, 1999; and U.S. patent application Ser. No.09/733,692, filed Dec. 8, 2000 (corresponding to InternationalPublication No. WO 01/42445A2), each incorporated herein by reference.

In other exemplary aspects of the invention, chimeric HPIV2 incorporatea HPIV2 vector genome or antigenome modified to express one or moremajor antigenic determinants of non-PIV pathogen, for example measlesvirus. The methods of the invention are generally adaptable forincorporation of antigenic determinants from a wide range of additionalpathogens within chimeric HPIV2 candidates. In this regard the inventionalso provides for development of candidates against subgroup A andsubgroup B respiratory syncytial viruses (RSV), HMPV, measles virus,human metapneumoviruses, mumps virus, human papilloma viruses, type 1and type 2 human immunodeficiency viruses, herpes simplex viruses,cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses,bunyaviruses, flaviviruses, alphaviruses and influenza viruses, amongother pathogens. Pathogens that may be targeted for development ofimmunogenic compositions according to the methods of the inventioninclude viral and bacterial pathogens, as well as protozoans andmulticellular pathogens. Useful antigenic determinants from manyimportant human pathogens in this context are known or readilyidentified for incorporation within chimeric HPIV2 of the invention.Thus, major antigens have been identified for the foregoing exemplarypathogens, including the measles virus HA and F proteins; the F, G, SHand M2 proteins of RSV, mumps virus HN and F proteins, human papillomavirus L1 protein, type 1 or type 2 human immunodeficiency virus gp160protein, herpes simplex virus and cytomegalovirus gB, gC, gD, gE, gG,gH, gI, gJ, gK, gL, and gM proteins, rabies virus G protein, humanmetapneuomovirus F and G proteins, Epstein Barr Virus gp350 protein;filovirus G protein, bunyavirus G protein, flavivirus E and NS1proteins, and alphavirus E protein. These major antigens, as well asother antigens known in the art for the enumerated pathogens and others,are well characterized to the extent that many of their antigenicdeterminants, including the full length proteins and their constituentantigenic domains, fragments and epitopes, are identified, mapped andcharacterized for their respective immunogenic activities.

Among the numerous, exemplary mapping studies that identify andcharacterize major antigens of diverse pathogens for use within theinvention are epitope mapping studies directed to thehemagglutinin-neuraminidase (HN) gene of HPIV3 (van Wyke Coelingh etal., J. Virol. 61:1473-1477, 1987, incorporated herein by reference).This report provides detailed antigenic structural analyses for 16antigenic variants of HPIV3 variants selected by using monoclonalantibodies (MAbs) to the HN protein that inhibit neuraminidase,hemagglutination, or both activities. Each variant possessed asingle-point mutation in the HN gene, coding for a single amino acidsubstitution in the HN protein. Operational and topographic maps of theHN protein correlated well with the relative positions of thesubstitutions. Computer-assisted analysis of the HN protein predicted asecondary structure composed primarily of hydrophobic sheetsinterconnected by random hydrophilic coil structures. The HN epitopeswere located in predicted coil regions. Epitopes recognized by MAbswhich inhibit neuraminidase activity of the virus were located in aregion which appears to be structurally conserved among severalparamyxovirus HN proteins and which may represent the sialicacid-binding site of the HN molecule.

This exemplary work, employing conventional antigenic mapping methods,identified single amino acids that are important for the integrity of HNepitopes. Most of these epitopes are located in the C-terminal half ofthe molecule, as expected for a protein anchored at its N terminus(Elango et al., J. Virol. 57:481-489, 1986). Previously publishedoperational and topographic maps of the PIV3 HN indicated that the MAbsemployed recognized six distinct groups of epitopes (I to VI) organizedinto two topographically separate sites (A and B), which are partiallybridged by a third site (C). These groups of epitopes represent usefulcandidates for antigenic determinants that may be incorporated, alone orin various combinations, within chimeric HPIV2 viruses of the invention.(See, also, Coelingh et al., Virology 143:569-582, 1985; Coelingh etal., Virology 162:137-143, 1988; Ray et al., Virology 148:232-236, 1986;Rydbeck et al., J. Gen. Virol. 67:1531-1542, 1986, each incorporatedherein by reference).

Additional studies by van Wyke Coelingh et al. (J. Virol. 63:375-382,1989) provide further information relating to selection of PIV antigenicdeterminants for use within the invention. In this study, twenty-sixmonoclonal antibodies (MAbs) (14 neutralizing and 12 nonneutralizing)were used to examine the antigenic structure, biological properties, andnatural variation of the fusion (F) glycoprotein of HPIV3. Analysis oflaboratory-selected antigenic variants and of PIV3 clinical isolatesindicated that the panel of MAbs recognizes at least 20 epitopes, 14 ofwhich participate in neutralization. Competitive binding assaysconfirmed that the 14 neutralization epitopes are organized into threenonoverlapping principal antigenic regions (A, B, and C) and one bridgesite (AB), and that the 6 nonneutralization epitopes form four sites (D,E, F, and G). Most of the neutralizing MAbs were involved innonreciprocal competitive binding reactions, suggesting that they induceconformational changes in other neutralization epitopes.

Other antigenic determinants for use within the invention have beenidentified and characterized for respiratory syncytial virus (RSV). Forexample, Beeler et al., J. Virol. 63:2941-2950, 1989, incorporatedherein by reference, employed eighteen neutralizing monoclonalantibodies (MAbs) specific for the fusion glycoprotein of the A2 strainof RSV to construct a detailed topological and operational map ofepitopes involved in RSV neutralization and fusion. Competitive bindingassays identified three nonoverlapping antigenic regions (A, B, and C)and one bridge site (AB). Thirteen MAb-resistant mutants (MARMs) wereselected, and the neutralization patterns of the MAbs with either MARMsor RSV clinical strains identified a minimum of 16 epitopes. MARMsselected with antibodies to six of the site A and AB epitopes displayeda small-plaque phenotype, which is consistent with an alteration in abiologically active region of the F molecule. Analysis of MARMs alsoindicated that these neutralization epitopes occupy topographicallydistinct but conformationally interdependent regions with uniquebiological and immunological properties. Antigenic variation in Fepitopes was then examined by using 23 clinical isolates (18 subgroup Aand 5 subgroup B) in cross-neutralization assays with the 18 anti-FMAbs. This analysis identified constant, variable, and hypervariableregions on the molecule and indicated that antigenic variation in theneutralization epitopes of the RSV F glycoprotein is the result of anoncumulative genetic heterogeneity. Of the 16 epitopes, 8 wereconserved on all or all but 1 of 23 subgroup A or subgroup B clinicalisolates. These antigenic determinants, including the full lengthproteins and their constituent antigenic domains, fragments andepitopes, all represent useful candidates for integration withinchimeric PIV of the invention to elicit novel immune responses asdescribed above. (See also, Anderson et al., J. Infect. Dis.151:626-633, 1985; Coelingh et al., J. Virol. 63:375-382, 1989; Fenneret al., Scand. J. Immunol. 24:335-340, 1986; Fernie et al., Proc. Soc.Exp. Biol. Med. 171:266-271, 1982; Sato et al., J. Gen. Virol.66:1397-1409, 1985; Walsh et al., J. Gen. Virol. 67:505-513, 1986, andOlmsted et al., J. Virol. 63:411-420, 1989, each incorporated herein byreference).

To express antigenic determinants of heterologous PIVs and non-PIVpathogens, the invention provides numerous methods and constructs. Incertain detailed embodiments, a transcription unit comprising an openreading frame (ORF) of a gene encoding an antigenic protein (e.g., themeasles virus HA gene) is added to a HPIV2 vector genome or antigenomeat various positions, yielding exemplary chimeric PIV1/measlescandidates. In exemplary embodiments, chimeric HPIV2 viruses areengineered that incorporate heterologous nucleotide sequences encodingprotective antigens from respiratory syncytial virus (RSV) to produceinfectious, attenuated candidates. The cloning of RSV cDNA and otherdisclosure pertaining to aspects of the invention set forth herein isprovided in U.S. patent application Ser. No. 08/720,132, filed Sep. 27,1996, corresponding to International Publication WO 97/12032 publishedApr. 3, 1997, and priority U.S. Provisional Patent Application No.60/007,083, filed Sep. 27, 1995; U.S. Provisional Patent Application No.60/021,773, filed Jul. 15, 1996; U.S. Provisional Patent Application No.60/046,141, filed May 9, 1997; U.S. Provisional Patent Application No.60/047,634, filed May 23, 1997; U.S. patent application Ser. No.08/892,403, filed Jul. 15, 1997 corresponding to InternationalPublication No. WO 98/02530 published on Jan. 22, 1998; U.S. patentapplication Ser. No. 09/291,894, filed on Apr. 13, 1999 corresponding toInternational Publication No. WO 00/61611 published Oct. 19, 2000, andpriority U.S. Provisional Patent Application Ser. No. 60/129,006, filedon Apr. 13, 1999; U.S. patent application Ser. No. 09/602,212, filedJun. 23, 2000 and corresponding International Publication No. WO01/04335 published on Jan. 18, 2001, and priority U.S. ProvisionalPatent Application Nos. 60/129,006, filed Apr. 13, 1999, 60/143,097,filed Jul. 9, 1999, and 60/143,132, filed Jul. 9, 1999; InternationalPublication No. WO 00/61737 published on Oct. 19, 2000; Collins et al.,Proc Nat. Acad. Sci. U.S.A. 92:11563-11567, 1995; Bukreyev et al., J.Virol. 70:6634-41, 1996, Juhasz et al., J. Virol. 71:5814-5819, 1997;Durbin et al., Virology 235:323-332, 1997; He et al. Virology237:249-260, 1997; Baron et al. J. Virol. 71:1265-1271, 1997; Whiteheadet al., Virology 247:232-9, 1998a; Whitehead et al., J. Virol.72:4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998; andWhitehead et al., J. Virol. 73:3438-3442, 1999, and Bukreyev et al.,Proc. Nat. Acad. Sci. U.S.A. 96:2367-72, 1999, each incorporated hereinby reference in its entirety for all purposes). Other reports anddiscussion incorporated or set forth herein identify and characterizeRSV antigenic determinants that are useful within the invention.

PIV chimeras incorporating one or more RSV antigenic determinants,preferably comprise a HPIV2 vector genome or antigenome combined with aheterologous gene or genome segment encoding an antigenic RSVglycoprotein, protein domain (e.g., a glycoprotein ectodomain) or one ormore immunogenic epitopes. In one embodiment, one or more genes orgenome segments from RSV F and/or G genes is/are combined with thevector genome or antigenome to form the chimeric HPIV2 candidate.Certain of these constructs will express chimeric proteins, for examplefusion proteins having a cytoplasmic tail and/or transmembrane domain ofHPIV2 fused to an ectodomain of RSV to yield a novel attenuated virusthat optionally elicits a multivalent immune response against both HPIV2and RSV.

Considering the epidemiology of RSV and HPIV1, HPIV2, and HPIV3, it maybe desired to administer immunogenic compositions of the invention in apredetermined, sequential schedule. RV and HPIV3 cause significantillness within the first four months of life whereas most of the illnesscaused by HPIV1 and HPIV2 occur after six months of age (Chanock et al.,in Parainfluenza Viruses, Knipe et al. (Eds.), pp. 1341-1379, LippincottWilliams & Wilkins, Philadelphia, 2001; Collins et al., In FieldsVirology, Vol. 1, pp. 1205-1243, Lippincott-Raven Publishers,Philadelphia, 1996; Reed et al., J. Infect. Dis. 175:807-13, 1997, eachincorporated herein by reference). Accordingly, certain sequentialimmunization protocols of the invention may involve administration of aimmunogenic composition as described herein that elicits an immuneresponse against HPIV3 and/or RSV (e.g., as a combined immunogeniccomposition) two or more times early in life, with the first doseadministered at or before one month of age, followed by an immunogeniccomposition against HPIV1 and/or HPIV2 at about four and six months ofage.

The invention therefore provides novel combinatorial immunogeniccompositions and coordinate immunization protocols for multiplepathogenic agents, including multiple PIVs and/or PIV and a non-PIVpathogen. These methods and formulations effectively target earlyimmunization against RSV and PIV3. One preferred immunization sequenceemploys one or more live attenuated immunogenic compositions against RSVand PIV3 as early as one month of age (e.g., at one and two months ofage) followed by a bivalent PIV1 and PIV2 immunogenic composition atfour and six months of age. It is thus desirable to employ the methodsof the invention to administer multiple PIV immunogenic compositions,including one or more chimeric PIV candidates, coordinately, e.g.,simultaneously in a mixture or separately in a defined temporal sequence(e.g., in a daily or weekly sequence), wherein each virus preferablyexpresses a different heterologous protective antigen. Such acoordinate/sequential immunization strategy, which is able to inducesecondary antibody responses to multiple viral respiratory pathogens,provides a highly powerful and extremely flexible immunization regimenthat is driven by the need to immunize against each of the three PIVviruses and other pathogens in early infancy.

Other sequential immunizations according to the invention permit theinduction of a high titer of antibody targeted to a heterologouspathogen, such as measles. In one embodiment, young infants (e.g. 2-4month old infants) are immunized with an attenuated HPIV3 or a chimericHPIV2-3 (incorporating HPIV3 antigenic determinant(s)s) and/or HPIV3virus that elicits an immune response against HPIV3 and/or aheterologous pathogen (for example a chimeric HPIV2 or HPIV3 virusexpressing the measles virus HA protein and also adapted to elicit animmune response against HPIV3). Subsequently, e.g., at four months ofage the infant is again immunized but with a different, secondary vectorconstruct, such as a recombinant HPIV2 virus expressing the measlesvirus HA gene and the HPIV2 antigenic determinants as functional,obligate glycoproteins of the vector. Following the first immunization,the recipient will elicit a primary antibody response to both the PIV3HN and F proteins and to the measles virus HA protein, but not to thePIV2 HN and F protein. Upon secondary immunization with the rHPIV2expressing the measles virus HA, the recipient will be readily infectedwith the immunogenic composition because of the absence of antibody tothe PIV2 HN and F proteins and will develop both a primary antibodyresponse to the PIV2 HN and F protective antigens and a high titeredsecondary antibody response to the heterologous measles virus HAprotein. A similar sequential immunization schedule can be developedwhere immunity is sequentially elicited against HPIV3 and then HPIV2 byone or more of the chimeric viruses disclosed herein, simultaneous withstimulation of an initial and then secondary, high titer immunogenicresponse against measles or another non-PIV pathogen. This sequentialimmunization strategy, preferably employing different serotypes of PIVas primary and secondary vectors, effectively circumvents immunity thatis induced to the primary vector, a factor ultimately limiting theusefulness of vectors with only one serotype. The success of sequentialimmunization with rHPIV3 and rHPIV3-1 virus candidates as describedabove has been reported (Tao et al., Vaccine 17:1100-8, 1999,incorporated herein by reference), but with the limitation of decreasedimmunogenicity of rHPIV3-1 against HPIV1 challenge. The presentinvention, in which the backbone of the booster virus is antigenicallyunrelated to the primary virus or vector, overcomes this importantlimitation.

Further in accordance with these aspects of the invention, exemplarycoordinate immunization protocols may incorporate two, three, four andup to six or more separate HPIV viruses administered simultaneously(e.g., in a polyspecific mixture) in a primary immunization step, e.g.,at one, two or four months of age. For example, two or more HPUV2-basedviruses can be administered that separately express one or moreantigenic determinants (i.e, whole antigens, immunogenic domains, orepitopes) selected from the G protein of RSV subgroup A, the F proteinof RSV subgroup A, the G protein of RSV subgroup B, the F protein of RSVsubgroup B, the G protein of HMPV, the F protein of HMPV, the HA proteinof measles virus, and/or the F protein of measles virus. Coordinatebooster administration of these same HPIV2-based constructs can berepeated at two months of age. Subsequently, e.g., at four months ofage, a separate panel of 2-6 or more antigenically distinct (referringto vector antigenic specificity) live attenuated HPIV2-based viruses canbe administered in a secondary immunization step. For example, secondaryimmunization may involve concurrent administration of a mixture ormultiple formulations that contain(s) multiple HPIV2 constructs thatcollectively express RSV G from subgroup A, RSV F from subgroup A, RSV Ffrom subgroup B, RSV G from subgroup B, measles virus HA, and/or measlesvirus F, or antigenic determinants from any combination of theseproteins. This secondary immunization provides a boost in immunity toeach of the heterologous RSV and measles virus proteins or antigenicdeterminant(s) thereof. At six months of age, a tertiary immunizationstep involving administration of one to six or more separate liveattenuated HPIV2-1 or HPIV2-3 vector-based recombinants can becoordinately administered that separately or collectively express RSV Gfrom subgroup A, RSV F from subgroup A, RSV G from subgroup B, RSV Ffrom subgroup B, the G protein of HMPV, the F protein of HMPV, measlesvirus HA, and/or measles virus F, or antigenic determinant(s) thereof.Optionally at this step in the immunization protocol, rHPIV3 and rHPIV2immunogenic compositions may be administered in booster formulations. Inthis way, the strong immunity characteristic of secondary antibody toHPIV1, HPIV2, HPIV3, RSV A, RSV B, HMPV, and measles viruses are allinduced within the first six months of infancy. Such acoordinate/sequential immunization strategy, which is able to inducesecondary antibody responses to multiple viral respiratory pathogens,provides a highly powerful and extremely flexible immunization regimenthat is driven by the need to immunize against each of the three PIVviruses and other pathogens in early infancy.

The present invention thus overcomes the difficulties inherent in priorapproaches to the development of vector based immunogenic compositionsand provides unique opportunities for immunization of infants during thefirst year of life against a variety of human pathogens. Previousstudies in developing live-attenuated PIV immunogenic compositionsindicate that, unexpectedly, rPIVs and their attenuated and chimericderivatives have properties that make them uniquely suited among thenonsegmented negative strand RNA viruses as vectors to express foreignproteins as immunogenic compositions against a variety of humanpathogens. The skilled artisan would not have predicted that the humanPIVs, which tend to grow substantially less well than the modelnonsegmented negative strand viruses and which typically have beenunderrepresented with regard to molecular studies, would prove to havecharacteristics which are highly favorable as vectors. It is alsosurprising that the intranasal route of administration of theseimmunogenic compositions has proven a very efficient means to stimulatea robust local and systemic immune response against both the vector andthe expressed heterologous antigen. Furthermore, this route providesadditional advantages for immunization against heterologous pathogensthat infect the respiratory tract or elsewhere.

The present invention provides major advantages over previous attemptsto immunize young infants against measles virus and other microbialpathogens. First, the HPIV2 recombinant vector into which the protectiveantigen or antigens of heterologous pathogens such as measles virus areinserted can be attenuated in a finely adjusted manner by incorporationof one or more attenuating mutations or other modifications to attenuatethe virus for the respiratory tract of the very young, seronegative orseropositive human infant. An extensive history of prior clinicalevaluation and practice (see, e.g., Karron et al., Pediatr. Infect. Dis.J. 15:650-654, 1996; Karron et al., J. Infect. Dis. 171:1107-1114,1995a; Karron et al., J. Infect. Dis. 172:1445-1450, 1995, eachincorporated herein by reference) greatly facilitates evaluation ofderivatives of these recombinants bearing foreign protective antigens inthe very young human infant.

Yet another advantage of the invention is that chimeric HPIV2 bearingheterologous sequences will replicate efficiently in vitro to enablelarge-scale production of immunogenic compositions. This is in contrastto the replication of some single-stranded, negative-sense RNA virusesthat can be inhibited in vitro by the insertion of a foreign gene(Bukreyev et al., J. Virol. 70:6634-41, 1996). Also, the presence ofthree antigenic serotypes of HPIV, each of which causes significantdisease in humans and hence can serve simultaneously as vector and toelicit an immune response, presents a unique opportunity to sequentiallyimmunize the infant with antigenically distinct variants of HPIV eachbearing the same foreign protein. In this manner the sequentialimmunization permits the development of a primary immune response to theforeign protein which can be boosted during subsequent infections withthe antigenically distinct HPIV also bearing the same or a differentforeign protein or proteins, i.e, the protective antigen of measlesvirus or of another microbial pathogen. It is also likely thatreadministration of homologous HPIV vectors will also boost the responseto both HPIV and the foreign antigen since the ability to cause multiplereinfections in humans is an unusual but characteristic attribute of theHPIVs (Chanock et al., Parainfluenza Viruses., p. 1341-1379, In D. M.Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B.Roizman, and S. E. Straus (eds.) Fields Virology, 4th ed., Vol. 1,Lippincott Williams & Wilkins, Philadelphia, 2001, incorporated hereinby reference).

Yet another advantage of the invention is that the introduction of agene unit into a HPIV2 vector has several highly desirable effects forthe production of attenuated viruses. First, the insertion of gene unitsexpressing, for example, the HA of measles virus or the HN of HPIV1 orHPIV3 can specify a host range phenotype on the HPIV2 vector, where theresulting HPIV2 vector replicates efficiently in vitro but is restrictedin replication in vivo in both the upper and lower respiratory tracts.Thus, the insertion of a gene unit expressing a viral protective antigenas an attenuating factor for the HPIV2 vector is a desirable property inlive attenuated virus of the invention.

The HPIV2 vector system has unique advantages over other members of thesingle-stranded, negative-sense viruses of the Order Mononegavirales.First, most other mononegaviruses that have been used as vectors are notderived from human pathogens (e.g., murine PIV1 (Sendai virus) (Sakai etal., FEBS Lett. 456:221-6, 1999), vesicular stomatitis virus (VSV) whichis a bovine pathogen (Roberts et al., J. Virol. 72:4704-11, 1998), andcanine PIV2 (SV5) He et al., Virology 237:249-60, 1997)). For thesenonhuman viruses, little or only weak immunity would be conferredagainst any human virus by antigens present in the vector backbone.Thus, a nonhuman virus vector expressing a supernumerary gene for ahuman pathogen would induce resistance only against that single humanpathogen. In addition, use of viruses such as SV5, rabies, or Sendaivirus as vector would expose subjects to viruses that they likely wouldnot otherwise encounter during life.

An important and specific advantage of the HPIV2 vector system is thatits preferred, intranasal route of administration, mimicking naturalinfection, will induce both mucosal and systemic immunity and reducesthe neutralizing and immunosuppressive effects of maternally-derivedserum IgG that is present in infants. While these same advantagestheoretically are possible for using RSV as a vector, for example, wehave found that RSV replication is strongly inhibited by inserts ofgreater than approximately 500 bp (Bukreyev et al., Proc. Natl. Acad.Sci. USA 96:2367-72, 1999). In contrast, as described herein, HPIV2 willoften accommodate several large gene inserts. The finding thatrecombinant RSV is unsuitable for bearing large inserts, whereasrecombinant PIVs are highly suitable, represents unexpected results.

It might be proposed that some other viral vector could be givenintranasally to obtain similar benefits as shown for PIV vectors, butthis has not been successful to date. For example, the MVA strain ofvaccinia virus expressing the protective antigens of HPIV3 was evaluatedas a live attenuated intranasal immunogenic composition against HPIV3.Although this vector appeared to be a very efficient expression systemin cell culture, it was inexplicably inefficient in inducing resistancein the upper respiratory tract of primates (Durbin et al., Vaccine16:1324-30, 1998, incorporated herein by reference) and was inexplicablyinefficient in inducing an effective immune response in the presence ofpassive serum antibodies (Durbin et al., J. Infect. Dis. 179:1345-51,1999, incorporated herein by reference). In contrast, PIV3 and RSVcandidates have been found to be protective in the upper and lowerrespiratory tract of non-human primates, even in the presence of passiveserum antibodies (Crowe et al., Vaccine 13:847-855, 1995; Durbin et al.,J. Infect. Dis. 179:1345-51, 1999, each incorporated herein byreference).

As noted above, the invention permits a wide range of alterations to berecombinantly produced within the HPIV2 genome or antigenome, yieldingdefined mutations that specify desired phenotypic changes. As also notedabove, defined mutations can be introduced by a variety of conventionaltechniques (e.g., site-directed mutagenesis) into a cDNA copy of thegenome or antigenome. The use of genomic or antigenomic cDNAsubfragments to assemble a complete genome or antigenome cDNA asdescribed herein has the advantage that each region can be manipulatedseparately, where small cDNA constructs provide for better ease ofmanipulation than large cDNA constructs, and then readily assembled intoa complete cDNA. Thus, the complete antigenome or genome cDNA, or aselected subfragment thereof, can be used as a template foroligonucleotide-directed mutagenesis. This can be through theintermediate of a single-stranded phagemid form, such as using theMUTA-gen® kit of Bio-Rad Laboratories (Richmond, Calif.), or a methodusing the double-stranded plasmid directly as a template such as theChameleon® mutagenesis kit of Strategene (La Jolla, Calif.), or by thepolymerase chain reaction employing either an oligonucleotide primer ora template which contains the mutation(s) of interest. A mutatedsubfragment can then be assembled into the complete antigenome or genomecDNA. A variety of other mutagenesis techniques are known and can beroutinely adapted for use in producing the mutations of interest in aPIV antigenome or genome cDNA of the invention.

Thus, in one illustrative embodiment mutations are introduced by usingthe MUTA-gene® phagemid in vitro mutagenesis kit available from Bio-RadLaboratories. In brief, cDNA encoding a PIV genome or antigenome iscloned into the plasmid pTZ18U, and used to transform CJ236 cells (LifeTechnologies). Phagemid preparations are prepared as recommended by themanufacturer. Oligonucleotides are designed for mutagenesis byintroduction of an altered nucleotide at the desired position of thegenome or antigenome. The plasmid containing the genetically alteredgenome or antigenome is then amplified.

Mutations can vary from single nucleotide changes to the introduction,deletion or replacement of large cDNA segments containing one or moregenes or genome segments. Genome segments can correspond to structuraland/or functional domains, e.g., cytoplasmic, transmembrane orectodomains of proteins, active sites such as sites that mediate bindingor other biochemical interactions with different proteins, epitopicsites, e.g., sites that stimulate antibody binding and/or humoral orcell mediated immune responses, etc. Useful genome segments in thisregard range from about 15-35 nucleotides in the case of genome segmentsencoding small functional domains of proteins, e.g., epitopic sites, toabout 50, 75, 100, 200-500, and 500-1,500 or more nucleotides.

The ability to introduce defined mutations into infectious recombinantHPIV2 has many applications, including the manipulation of PIVpathogenic and immunogenic mechanisms. For example, the functions ofHPIV2 proteins, including the N, P, M, F, HN, and L proteins andproducts of the V ORF, can be manipulated by introducing mutations whichablate or reduce the level of protein expression, or which yield mutantprotein. Various genome RNA structural features, such as promoters,intergenic regions, and transcription signals, can also be routinelymanipulated within the methods and compositions of the invention. Theeffects of trans-acting proteins and cis-acting RNA sequences can bereadily determined, for example, using a complete antigenome cDNA inparallel assays employing PIV minigenomes (Dimock et al., J. Virol.67:2772-8, 1993, incorporated herein by reference in its entirety),whose rescue-dependent status is useful in characterizing those mutantsthat may be too inhibitory to be recovered in replication-independentinfectious virus.

Certain substitutions, insertions, deletions or rearrangements of genesor genome segments within recombinant HPIV2 of the invention (e.g.,substitutions of a genome segment encoding a selected protein or proteinregion, for instance a cytoplasmic tail, transmembrane domain orectodomain, an epitopic site or region, a binding site or region, anactive site or region containing an active site, etc.) are made instructural or functional relation to an existing, “counterpart” gene orgenome segment from the same or different PIV or other source. Suchmodifications yield novel recombinants having desired phenotypic changescompared to wild-type or parental PIV or other viral strains. Forexample, recombinants of this type may express a chimeric protein havinga cytoplasmic tail and/or transmembrane domain of one PIV fused to anectodomain of another PIV. Other exemplary recombinants of this typeexpress duplicate protein regions, such as duplicate immunogenicregions.

As used herein, “counterpart” genes, genome segments, proteins orprotein regions, are typically from heterologous sources (e.g., fromdifferent PIV genes, or representing the same (i.e, homologous orallelic) gene or genome segment in different PIV types or strains).Typical counterparts selected in this context share gross structuralfeatures, e.g., each counterpart may encode a comparable protein orprotein structural domain, such as a cytoplasmic domain, transmembranedomain, ectodomain, binding site or region, epitopic site or region,etc. Counterpart domains and their encoding genome segments embrace anassemblage of species having a range of size and sequence variationsdefined by a common biological activity among the domain or genomesegment variants.

Counterpart genes and genome segments, as well as other polynucleotidesdisclosed herein for producing recombinant PIV within the invention,often share substantial sequence identity with a selected polynucleotide“reference sequence,” e.g., with another selected counterpart sequence.As used herein, a “reference sequence” is a defined sequence used as abasis for sequence comparison, for example, a segment of a full-lengthcDNA or gene, or a complete cDNA or gene sequence. Generally, areference sequence is at least 20 nucleotides in length, frequently atleast 25 nucleotides in length, and often at least 50 nucleotides inlength. Since two polynucleotides may each (1) comprise a sequence (i.e,a portion of the complete polynucleotide sequence) that is similarbetween the two polynucleotides, and (2) may further comprise a sequencethat is divergent between the two polynucleotides, sequence comparisonsbetween two (or more) polynucleotides are typically performed bycomparing sequences of the two polynucleotides over a “comparisonwindow” to identify and compare local regions of sequence similarity. A“comparison window”, as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e, gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith & Waterman, (Adv. Appl. Math. 2:482, 1981), by the homologyalignment algorithm of Needleman & Wunsch, (J. Mol. Biol. 48:443, 1970),by the search for similarity method of Pearson & Lipman, (Proc. Natl.Acad. Sci. U.S.A. 85:2444, 1988) (each incorporated herein byreference), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.,incorporated herein by reference), or by inspection, and the bestalignment (i.e, resulting in the highest percentage of sequencesimilarity over the comparison window) generated by the various methodsis selected. The term “sequence identity” means that two polynucleotidesequences are identical (i.e, on a nucleotide-by-nucleotide basis) overthe window of comparison. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, U, or 1) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e, the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The terms “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 85 percent sequence identity, preferably at least 90 to 95 percentsequence identity, more usually at least 99 percent sequence identity ascompared to a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence.

In addition to these polynucleotide sequence relationships, proteins andprotein regions encoded by recombinant HPIV2 of the invention are alsotypically selected to have conservative relationships, i.e to havesubstantial sequence identity or sequence similarity, with selectedreference polypeptides. As applied to polypeptides, the term “sequenceidentity” means peptides share identical amino acids at correspondingpositions. The term “sequence similarity” means peptides have identicalor similar amino acids (i.e, conservative substitutions) atcorresponding positions. The term “substantial sequence identity” meansthat two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 90 percent sequenceidentity, more preferably at least 95 percent sequence identity or more(e.g., 99 percent sequence identity). The term “substantial similarity”means that two peptide sequences share corresponding percentages ofsequence similarity. Preferably, residue positions that are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Abbreviations for the twenty naturally occurringamino acids used herein follow conventional usage (Immunology—ASynthesis, 2nd ed., E. S. Golub & D. R. Gren, eds., Sinauer Associates,Sunderland, Mass., 1991, incorporated herein by reference).Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as a, a-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, w-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). Moreover,amino acids may be modified by glycosylation, phosphorylation and thelike.

To select candidate viruses according to the invention, the criteria ofviability, attenuation and immunogenicity are determined according towell-known methods. Viruses that will be most desired in immunogeniccompositions of the invention must maintain viability, have a stableattenuation phenotype, exhibit replication in an immunized host (albeitat lower levels), and effectively elicit production of an immuneresponse in a recipient sufficient to elicit a desired immune response.The recombinant HPIV2 viruses of the invention are not only viable andappropriately attenuated, they are more stable genetically invivo—retaining the ability to stimulate an immune response and in someinstances to expand the immune response elicited by multiplemodifications, e.g., induce an immune response against different viralstrains or subgroups, or to stimulate a response mediated by a differentimmunologic basis, e.g., secretory versus serum immunoglobulins,cellular immunity, and the like.

Recombinant HPIV2 viruses of the invention can be tested in variouswell-known and generally accepted in vitro and in vivo models to confirmadequate attenuation, resistance to phenotypic reversion, andimmunogenicity. In in vitro assays, the modified virus (e.g., a multiplyattenuated, biologically derived or recombinant PIV) is tested, e.g.,for temperature sensitivity of virus replication, i.e ts phenotype, andfor the small plaque or other desired phenotype. Modified viruses arefurther tested in animal models of PIV infection. A variety of animalmodels have been described and are summarized in various referencesincorporated herein. PIV model systems, including rodents and non-humanprimates, for evaluating attenuation and immunogenic activity of PIVcandidates are widely accepted in the art, and the data obtainedtherefrom correlate well with PIV infection, attenuation andimmunogenicity in humans.

In accordance with the foregoing description, the invention alsoprovides isolated, infectious recombinant HPIV2 for use in immunogeniccompositions. The attenuated virus which is a component of animmunogenic composition is in an isolated and typically purified form.By isolated is meant to refer to PIV which is in other than a nativeenvironment of a wild-type virus, such as the nasopharynx of an infectedindividual. More generally, isolated is meant to include the attenuatedvirus as a component of a cell culture or other artificial medium whereit can be propagated and characterized in a controlled setting. Forexample, attenuated HPIV2 of the invention may be produced by aninfected cell culture, separated from the cell culture and added to astabilizer.

For use in immunogenic compositions, recombinant HPIV2 producedaccording to the present invention can be used directly in formulations,or lyophilized, as desired, using lyophilization protocols well known tothe artisan. Lyophilized virus will typically be maintained at about 4°C. When ready for use the lyophilized virus is reconstituted in astabilizing solution, e.g., saline or comprising SPG, Mg⁺⁺ and HEPES,with or without adjuvant, as further described below.

HPIV2-based immunogenic compositions of the invention contain as anactive ingredient an immunogenically effective amount of a recombinantHPIV2 produced as described herein. The modified virus may be introducedinto a host with a physiologically acceptable carrier and/or adjuvant.Useful carriers are well known in the art, and include, e.g., water,buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.The resulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration, as mentioned above. The compositionsmay contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions, such as pH adjusting andbuffering agents, tonicity adjusting agents, wetting agents and thelike, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, and the like. Acceptable adjuvants includeincomplete Freund's adjuvant, MPL™ (3-0-deacylated monophosphoryl lipidA; Corixa, Hamilton Ind.) and IL-12 (Genetics Institute, CambridgeMass.), among many other suitable adjuvants well known in the art.

Upon immunization with a recombinant HPIV2 composition as describedherein, via aerosol, droplet, oral, topical or other route, the immunesystem of the host responds to the immunogenic composition by producingantibodies specific for PIV proteins, e.g., F and HN glycoproteins. As aresult of the immunization with an immunogenically effective amount of arecombinant HPIV2 produced as described herein, the host becomes atleast partially or completely immune to infection by the targeted PIV ornon-PIV pathogen, or resistant to developing moderate or severeinfection therefrom, particularly of the lower respiratory tract.

The host to which the immunogenic compositions are administered can beany mammal which is susceptible to infection by PIV or a selectednon-PIV pathogen and which host is capable of generating an immuneresponse to the antigens of the immunizing strain. Accordingly, theinvention provides methods for creating immunogenic compositions for avariety of human and veterinary uses.

The compositions containing the recombinant HPIV2 of the invention areadministered to a host susceptible to or otherwise at risk for PIVinfection to enhance the host's own immune response capabilities. Suchan amount is defined to be a “immunogenically effective dose.” In thisuse, the precise amount of recombinant HPIV2 to be administered withinan effective dose will depend on the host's state of health and weight,the mode of administration, the nature of the formulation, etc., butwill generally range from about 10³ to about 10⁷ plaque forming units(PFU) or more of virus per host, more commonly from about 10⁴ to 10⁶ PFUvirus per host. In any event, the formulations should provide a quantityof modified PIV of the invention sufficient to elicit a detectableimmune response in the host patient against the subject pathogen(s).

The recombinant HPIV2 produced in accordance with the present inventioncan be combined with viruses of other PIV serotypes or strains to elicita desired immune response against multiple PIV serotypes or strains.Alternatively, an immune response against multiple PIV serotypes orstrains can be achieved by combining protective epitopes of multipleserotypes or strains engineered into one virus, as described herein.Typically when different viruses are administered they will be inadmixture and administered simultaneously, but they may also beadministered separately. Immunization with one strain may immunizeagainst different strains of the same or different serotype.

In some instances it may be desirable to combine the recombinant HPIV2immunogenic compositions of the invention with immunogenic compositionsthat induce immune responses to other agents, particularly otherchildhood viruses. In another aspect of the invention the recombinantHPIV2 can be employed as a vector for protective antigens of otherpathogens, such as respiratory syncytial virus (RSV) or measles virus,by incorporating the sequences encoding those protective antigens intothe recombinant HPIV2 genome or antigenome that is used to produceinfectious virus, as described herein.

In all subjects, the precise amount of recombinant HPIV2 administered,and the timing and repetition of administration, will be determinedusing conventional methods based on the patient's state of health andweight, the mode of administration, the nature of the formulation, etc.Dosages will generally range from about 10³ to about 10⁷ plaque formingunits (PFU) or more of virus per patient, more commonly from about 10⁴to 10⁶ PFU virus per patient. In any event, the formulations shouldprovide a quantity of attenuated recombinant HPIV2 sufficient toeffectively stimulate or induce an anti-PIV or other anti-pathogenicimmune response, e.g., as can be determined by hemagglutinationinhibition, complement fixation, plaque neutralization, and/orenzyme-linked immunosorbent assay, among other methods. In this regard,individuals are also monitored for signs and symptoms of upperrespiratory illness. As with administration to chimpanzees, theattenuated virus of grows in the nasopharynx of recipients at levelsapproximately 10-fold or more lower than wild-type virus, orapproximately 10-fold or more lower when compared to levels ofincompletely attenuated virus.

In neonates and infants, multiple administrations may be required toelicit sufficient levels of immunity. Administration should begin withinthe first month of life, and at intervals throughout childhood, such asat two months, six months, one year and two years, as necessary tomaintain an immune response against native (wild-type) PIV infection.Similarly, adults who are particularly susceptible to repeated orserious PIV infection, such as, for example, health care workers, daycare workers, family members of young children, the elderly, individualswith compromised cardiopulmonary function, may require multipleimmunizations to establish and/or maintain immune responses. Levels ofinduced immunity can be monitored by measuring amounts of neutralizingsecretory and serum antibodies, and dosages adjusted or immunizationsrepeated as necessary to maintain desired levels of immune response.Further, different candidate viruses may be indicated for administrationto different recipient groups. For example, an engineered HPIV2expressing a cytokine or an additional protein rich in T cell epitopesmay be particularly advantageous for adults rather than for infants.

HPIV2-based immunogenic compositions produced in accordance with thepresent invention can be combined with viruses expressing antigens ofanother subgroup or strain of PIV to elicit an immune response againstmultiple PIV subgroups or strains. Alternatively, the candidate virusmay incorporate protective epitopes of multiple PIV strains or subgroupsengineered into one PIV clone, as described herein.

The recombinant HPIV2 immunogenic compositions of the invention elicitproduction of an immune response that reduces or alleviates seriouslower respiratory tract disease, such as pneumonia and bronchiolitiswhen the individual is subsequently infected with wild-type PIV. Whilethe naturally circulating virus is still capable of causing infection,particularly in the upper respiratory tract, there is a very greatlyreduced possibility of rhinitis as a result of the immunization.Boosting of resistance by subsequent infection by wild-type virus canoccur. Following immunization, there are detectable levels of hostengendered serum and secretory antibodies which are capable ofneutralizing homologous (of the same subgroup) wild-type virus in vitroand in vivo.

Preferred recombinant HPIV2 candidates of the invention exhibit a verysubstantial diminution of virulence when compared to wild-type virusthat naturally infects humans. The virus is sufficiently attenuated sothat symptoms of infection will not occur in most immunized individuals.In some instances the attenuated virus may still be capable ofdissemination to unimmunized individuals. However, its virulence issufficiently abrogated such that severe lower respiratory tractinfections in the immunized host do not occur.

The level of attenuation of recombinant HPIV2 candidates may bedetermined by, for example, quantifying the amount of virus present inthe respiratory tract of an immunized host and comparing the amount tothat produced by wild-type PIV or other attenuated PIV which have beenevaluated as candidate strains. For example, the attenuated virus of theinvention will have a greater degree of restriction of replication inthe upper respiratory tract of a highly susceptible host, such as achimpanzee, compared to the levels of replication of wild-type virus,e.g., 10- to 1000-fold less. In order to further reduce the developmentof rhinorrhea, which is associated with the replication of virus in theupper respiratory tract, a useful candidate virus should exhibit arestricted level of replication in both the upper and lower respiratorytract. However, the attenuated viruses of the invention must besufficiently infectious and immunogenic in humans to elicit a desiredimmune response in immunized individuals. Methods for determining levelsof PIV in the nasopharynx of an infected host are well known in theliterature.

Levels of induced immunity provided by the immunogenic compositions ofthe invention can also be monitored by measuring amounts of neutralizingsecretory and serum antibodies. Based on these measurements, dosages canbe adjusted or immunizations repeated as necessary to maintain desiredlevels of immune response. Further, different candidate viruses may beadvantageous for different recipient groups. For example, an engineeredrecombinant HPIV2 strain expressing an additional protein rich in T cellepitopes may be particularly advantageous for adults rather than forinfants.

In yet another aspect of the invention the recombinant HPIV2 is employedas a vector for transient gene therapy of the respiratory tract.According to this embodiment the recombinant HPIV2 genome or antigenomeincorporates a sequence that is capable of encoding a gene product ofinterest. The gene product of interest is under control of the same or adifferent promoter from that which controls PIV expression. Theinfectious recombinant HPIV2 produced by coexpressing the recombinantHPIV2 genome or antigenome with the N, P, L and other desired PIVproteins, and containing a sequence encoding the gene product ofinterest, is administered to a patient. Administration is typically byaerosol, nebulizer, or other topical application to the respiratorytract of the patient being treated. Recombinant HPIV2 is administered inan amount sufficient to result in the expression of therapeutic orprophylactic levels of the desired gene product. Representative geneproducts that may be administered within this method are preferablysuitable for transient expression, including, for example,interleukin-2, interleukin-4, gamma-interferon, GM-CSF, G-C SF,erythropoietin, and other cytokines, glucocerebrosidase, phenylalaninehydroxylase, cystic fibrosis transmembrane conductance regulator (CFTR),hypoxanthine-guanine phosphoribosyl transferase, cytotoxins, tumorsuppressor genes, antisense RNAs, and other candidate antigens.

The following examples are provided by way of illustration, notlimitation. These examples describe the development of a novel reversegenetics system for the recovery of HPIV2 from cDNA, and the use of thissystem for construction of novel recombinant HPIV2 candidates. Briefly,the examples below detail investigations leading to the completesequence of a clinical isolate of HPIV2. Also described is theconstruction of a complete antigenomic cDNA, rescue of infectious,recombinant HPIV2 virus, and investigations to characterize thephenotype of recombinant HPIV2 candidates of the invention in vitro andin vivo.

EXAMPLE I Sequence Determination of Human Parainfluenza Virus Type 2(HPIV2) and Generation of a Recombinant Wild Type HPIV2 from Cloned DNA

The present Example demonstrates complete genomic sequence determinationof a human parainfluenza virus type 2 (HPIV2) virus that can be directlyrecovered, without modification into recombinantly derived HPIV2. Thesubject viral strain for this analysis is Vanderbilt/1994 (V94), whichwas originally isolated in 1994 from an infected 13 month-old infant.The HPIV2/V94 genome is shown herein to be 15,654 nucleotides in lengthand, therefore, to conform to the “rule of six”. This rule relates tothe observation that efficient RNA replication by members of subfamilyParamyxovirinae, family Paramyxoviridae, requires a genome nucleotidelength that is evenly divisible by six (Kolakofsky et al., J. Virol.72:891-9, 1998, incorporated herein by reference).

The determination of a complete wt HPIV2 sequence disclosed hereinallows the generation of cDNAs that can be used to produce HPIV2 ofdefined sequence and growth characteristics. In the present example, awt rHPIV2/V94 cDNA is described that can be used as a substrate forsystematically introducing attenuating mutations to derive liveattenuated HPIV2 candidates, whose genome length and sequence are welldefined. In addition, genes encoding foreign protective antigens can beintroduced into the rHPIV2/V94 genome to produce live-attenuatedcandidates able to elicit an immune response against HPIV2 and otherpathogens. These attenuated HPIV2 vectors will typically be designed tohave genome lengths that conform to the rule of six.

Cell Lines and Viruses

HEp-2 (ATCC CCL 23) and LLC-MK2 (ATCC CCL 7.1) cells were maintained inOptiMEM I (Life Technologies, Gaithersburg, Md.) supplemented with 5%FBS and gentamicin sulfate (50 ug/mL). Recombinant and biologicallyderived HPIV2s were propagated in LLC-MK2 cells and were quantified bylimiting dilution with virus-infected cultures identified byhemadsorption with guinea pig erythrocytes, as described previously(Hall et al., Virus Res. 22:173-184, 1992, incorporated herein byreference).

Virion RNA Isolation

Confluent monolayers of LLC-MK2 cells were infected with HPIV2/V94 at amultiplicity of infection (m.o.i.) of approximately one TCID₅₀ per cell.At 4-6 days post-infection, clarified supernatants were harvested andvirus was precipitated by incubation in 7.5% (w/v) PEG-8000 on ice for 2hr followed by centrifugation at 10,845×g for 1 hr. Virion RNA (vRNA)was isolated by extraction of the pellet with TRIzol reagent(Invitrogen, Inc., Carlsbad, Calif.) and chloroform. vRNA wasprecipitated with an equal volume of isopropanol. The vRNA pellet waswashed in 70% ethanol and resuspended in diethyl pyrocarbonate (DEPC)treated H₂O.

Reverse Transcription (RT), Polymerase Chain Reaction (PCR), andNucleotide Sequencing

vRNA was reverse transcribed using the Thermoscript RT-PCR System(Invitrogen, Inc.) and random hexamer primers. PCR was carried out onthe reverse transcribed cDNA product using the Herculase EnhancedPolymerase Blend (Stratagene, LaJolla, Calif.). The antigenomic HPIV2cDNA was generated from the RT and RACE products (see below) in sixoverlapping PCR fragments using primers homologous to fragments frompreviously published strains of HPIV2, or primers based on HPIV2/V94sequence obtained during the course of the experiments. The nucleotidesequences of cDNA products were determined by direct sequence analysisof the RT-PCR products using a Perkin-Elmer ABI 3100 sequencer with thedRhodamine sequencing kit (Perkin-Elmer Applied Biosystems, Warrington,UK). The sequence was assembled from the six overlapping RT-PCRfragments using the Autoassembler (Perkin-Elmer Applied Biosystems)program.

The 3′ terminal genomic sequence of HPIV2 was converted to cDNA usingthe 3′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen,Inc.) as specified by the manufacturer. Briefly, vRNA was polyadenylatedat its 3′-end using poly A polymerase (Invitrogen. Inc.) followed byfirst-strand cDNA synthesis primed with oligo (dT) and PCR using anHPIV2 specific reverse primer and a forward AUAP primer supplied withthe kit. RACE products were sequenced directly as described above. Todetermine the sequence for the 3′-end, two independently derived RACEproducts were sequenced and were found to be identical.

The 5′ genomic terminus of HPIV2 was amplified from vRNA followingfirst-strand cDNA synthesis, terminal transferase tailing, and PCRamplification as specified by the 5′ RACE System for Rapid Amplificationof cDNA 5′ end Version 2.0 (Invitrogen, Inc.). The amplified cDNA RACEproducts were sequenced directly. The sequence for the 5′-end wasdetermined with multiple sequencing reactions of two independentlyderived 5′ RACE products, and the sequences were found to be identical.

Assembly of a Full-Length rHPIV2 cDNA Antigenomic Clone

A full-length cDNA clone encoding the HPIV2 antigenomic RNA wasconstructed from six cloned overlapping RT-PCR and RACE products usingthe following restriction sites in the HPIV2 genome: Nhe I (nt 892position in the complete antigenomic sequence), Xho I (nt 3673), Asp 718(nt 8141), Aat II (nt 10342), and Bsi WI (nt 15280) (FIG. 3). Briefly,the first fragment was generated using the 3′ RACE system (Invitrogen,Inc.) and the Herculase Enhanced Polymerase Blend (Stratagene, La Jolla,Calif.) with specific HPIV2 primers and a primer containing an Mlu Isite followed by a T7 promoter, two non-viral G residues, and theantigenomic cDNA corresponding to 3′-end of the viral genome. The nextfour fragments (FIG. 3) were reverse transcribed using the ThermoscriptRT-PCR System (Invitrogen, Inc.) and PCR amplified with the HerculaseEnhanced Polymerase Blend (Stratagene). The final fragment containingthe 5′-end was reverse transcribed using the 5′ RACE System (Invitrogen,Inc.) and amplified with the Herculase Enhanced Polymerase Blend(Stratagene). The individual PCR products were cloned into a pUC19plasmid modified to contain restriction enzyme sites to accept the PCRfragments, and were sequenced using a Perkin-Elmer ABI 3100 sequencerwith the BigDye sequencing kit (Perkin-Elmer Applied Biosystems), andcompared to the consensus sequence of HPIV2/V94 prior to assembly.

All six PCR products were assembled into a pUC19 vector modified tocontain Mlu I, BspEI, Xho I, Asp 718, Aat II, Bsi WI, Cla I and Rsr IIrestriction sites to generate pUC-HPIV2/V94. The infectious full-lengthcDNA plasmid, designated pFLC-HPIV2/V94, was generated by subcloning theMlu I to Rsr II HPIV2/V94 cDNA fragment from pUC-HPIV2/V94 into amodified pBlueScript (Schmidt et al., J. Virol. 74:8922-9, 2000,incorporated herein by reference), and contains the following elements:a T7 promoter followed by two nonviral guanosine residues, the completeantigenomic 15654 nt sequence of HPIV2/V94, a hepatitis delta virusribozyme, and a T7 polymerase transcriptional terminator as previouslydescribed for the generation of recombinant HPIV3, BPIV3, and HPIV1(Durbin et al., Virology 235:323-332, 1997; Schmidt et al., J. Virol.74:8922-9, 2000, each incorporated herein by reference). The sequence ofthe full-length cDNA was verified by DNA sequence analysis. Two pointmutations were identified in the F and L ORFs of the full-length cDNAthat had not been present in the consensus sequence occurred during theassembly of the clone at positions 6265 (agt to agc; silent) and 15075(gct to gtt; Ala-2093 to Val). These mutations were used as markers todistinguish the recombinant HPIV2 from the biologically derivedHPIV2/V94 parent.

HPIV2 N, P, and L Support Plasmids for the Recovery of HPIV2/V94 fromcDNA

A support plasmid encoding the N protein of HPIV2/V94 (pTM-N₂) wasderived from vRNA using the Thermoscript RT-PCR System (Invitrogen,Inc.) and the Advantage-HF PCR kit (Clontech) using a antigenomic senseoligonucleotide that contained an Afl III site spanning the N ORF ATGtranslation initiation codon site and an anti-sense oligo containing anEcoRI site. The PCR product was digested with Afl III and EcoRI andcloned into pTM1 (Durbin et al., Virology 235:323-332, 1997; Durbin etal., Virology 234:74-83, 1997; Elroy-Stein et al., Proc. Natl. Acad.Sci. USA. 86:6126-30, 1989, each incorporated herein by reference) thatwas digested with Nco I and EcoRI.

The HPIV2/V94 P protein expression plasmid (pTM-P₂)) was generated fromtwo overlapping PCR fragments (Moeller et al., J. Virol. 75:7612-20,2001, incorporated herein by reference) and was engineered to contain atwo guanosine nt insertion within the HPIV2 P gene editing site (nt2481-2487), to generate the complete P ORF (as distinguished therebyfrom the V ORF), which was subcloned into pTM 1 as an Nco I to EcoRIfragment.

An HPIV2 L polymerase expression plasmid (pTM-L₂) was made by PCRamplification with a sense oligo containing an Nco I site spanning the Lgene ATG translation initiation codon, and an antisense oligo downstreamof a unique Aat II site (nt 10342) in the L ORF. The remainder of the LORF was derived from a subclone used to construct the HPIV2 full-lengthclone. The PCR product was digested with Asp718 and Aat II and wascloned into a pUC19 plasmid containing the HPIV2 nts 10342 to 15654followed by the unique extragenomic Rsr II site. The complete HPIV2/V94L ORF was then subcloned into a modified pTM1 as an Nco I to Rsr IIfragment.

Recovery of Viruses from cDNA and Sequencing of Viral RNA (vRNA)

Recombinant HPIV2/V94 was recovered from two independent clones of thefull length HPIV2/V94 antigenomic cDNA transfected into HEp-2 cells, asdescribed previously (Skiadopoulos et al., Virology 272:225-34, 2000,incorporated herein by reference). Briefly, HEp-2 cells in 6-well plates(Costar, Corning, Inc., Corning, N.Y.) were co-transfected with afull-length HPIV2N94 cDNA plasmid and the three HPIV2 support plasmids(pTM N₂, pTM P₂, pTM L₂), using Lipofectamine-2000 reagent (Invitrogen,Inc.). The HEp-2 cells were simultaneously infected with MVA-T7 asdescribed previously (Durbin et al., Virology 235:323-332, 1997; Schmidtet al., J. Virol. 74:8922-9, 2000, each incorporated herein byreference). Supernatant was harvested on day three or fourpost-transfection and was passaged two times on LLC-MK2 monolayers. Toconfirm that viruses were derived from cDNA rather than representingcontamination by biologically derived virus, RT was performed andsegments of the viral genome were amplified by PCR. Sequence analysis ofthe PCR products revealed the presence of the two point mutations thatare present in the F and L genes of the recombinant virus, designatedrHPIV2/V94, but that are not present in the wild type parental virus. Asa control, PCR was performed without the prior RT step: this did notyield a detectable product, showing that the template was RNA ratherthan containing DNA. Each rHPIV2/V94 was then biologically-cloned byplaque to plaque purification on LLC-MK2 monolayers and was furtherpropagated on LLC-MK2 cells, as previously described (Skiadopoulos etal., Virology 260:125-35, 1999, incorporated herein by reference) togenerate two pools of HPIV2/V94 (designated r_(A)HPIV2/V94 andr_(B)HPIV2/V94) derived from two independent transfections.

vRNA was isolated from the biologically cloned r_(A)HPIV2/V94 asdescribed previously (Skiadopoulos et al., Virology 260:125-35, 1999,incorporated herein by reference) and was used as the template forreverse transcription and polymerase chain reaction (RT-PCR) usingoligonucleotide primers. The amplified products were analyzed by DNAsequencing to determine the complete genomic sequence of r_(A)HPIV2/V94.

Results

The complete genomic sequence of HPIV2 strain Vanderbilt 1994(HPIV2/V94) (sequence provided in FIGS. 9A-F; GenBank accession no.AF533010, incorporated herein by reference) was determined from RT-PCRproducts amplified from purified vRNA. The sequence analysis wasperformed directly on RT-PCR products without a cloning step, and thusyielded a reliable consensus sequence. The HPIV2 genome was determinedto be 15,654 bp in length, and thus the sequence provided hereinconforms to the rule of six.

A complete HPIV2 antigenomic cDNA, designated pFLC-HPIV2/V94, wasconstructed from six overlapping RT-PCR and RACE products and containedtwo nucleotide changes in the F and L genes, respectively, as markers(FIG. 3). The antigenomic cDNA was transfected into HEp-2 cells andvirus was recovered using the support HPIV2/V94 N, P, and L proteinexpression plasmids and co-infection with MVA-17. Virus was readilyrecovered after only a single passage of the HEp-2 transfectionsupernatant onto LLC-MK2 monkey kidney cell monolayers, and the presenceof the nucleotide markers present in the HPIV2 F and L genes wasconfirmed by RT-PCR of vRNA and sequence analysis. Amplification ofRT-PCR products was dependent on the addition of RT, indicating that thetemplate was indeed viral RNA and not contaminating DNA.

The complete sequence of r_(A)HPIV2/V94 was determined from RT-PCRproducts of vRNA isolated from recombinant virus that had beenbiologically cloned by sequential plaque to plaque purification.Specifically, the isolated genomic sequence of the recovered virus wasidentical to that of the biologically derived HPIV2 strain V94 parentexcept for two incidental, single-nt substitution mutations that werepresent in the antigenomic cDNA and serve as markers for recombinantHPIV2. The first mutation at position 6265 (T to C) in the F gene istranslationally silent. The second mutation at nt 15075 (C to T) resultsin an alanine-2093 to valine substitution in the L gene. This amino acidposition is not conserved between other parainfluenza viruses and liesoutside of the six domains that are conserved between Paramyxoviruses.These point mutations serve as markers for a virus derived from cDNA. Athird translationally silent nt substitution at nt 13786 (T to C) thatarose during propagation of the virus in cell culture was alsoidentified. The incidental mutations did not alter growth in vitro (seebelow). Partial sequence analysis of r_(B)HPIV2 showed that thisincidental mutation was not present, consistent with the idea that it isan inconsequential mutation acquired during virus growth.

EXAMPLE II Replication of Recombinant HPIV2 In Vitro

The mean peak titer of recombinant and biologically derived HPIV2 wasdetermined by multi-cycle growth titrations. Each HPIV2 tested wasinoculated in triplicate into LLC-MK2 monolayers in 6-well plates at amultiplicity of infection (m.o.i.) of 0.01, and cultures were incubatedat 32° C. with and without porcine derived trypsin added to the culturemedium, as described previously (16). 0.5 ml of medium from each wellwas harvested and replaced with 0.5 ml of fresh medium at 0 hr and at 1to 7 days post-infection. Virus present in the samples was quantified bytitration on LLC-MK2 monolayers in 96-well plates that were incubatedfor seven days at 32° C. Virus was detected by hemadsorption and isreported as log₁₀ TCID₅₀/ml (50% tissue culture infectious dose/ml).

The growth properties in cell culture of rHPIV2/V94 recovered from cDNAwere indistinguishable from that of the biologically derived HPIV2/V94(FIG. 4), and growth in vitro did not require the addition of trypsin tothe culture medium (compare FIG. 4, panels A and B). Growth at 32° C.,39° C. and 40° C. was also examined, and the rHPIV2/V94 and biologicallyderived viruses were not ts at the higher temperatures. Thus, theincidental nt substitutions in rHPIV2/V94 did not alter growthproperties in vitro.

EXAMPLE III Replication of rHPIV2/V94 and Biologically Derived Wild TypeHPIV2/V94 in Hamsters

4 week-old Golden Syrian hamsters (Charles River Laboratories, NY), ingroups of 6 were inoculated intranasally (IN) with 0.1 ml of L15 mediumcontaining 10^(6.0) TCID₅₀ of virus. On day 3, 4 or 5 post-infection,the lungs and nasal turbinates were harvested, and the virus wasquantified by serial dilution of tissue homogenates on LLC-MK2monolayers, as previously described (Skiadopoulos et al., J. Virol.72:1762-8, 1998, incorporated herein by reference). The mean virus titerwas calculated for each group of hamsters and is expressed as log₁₀TCID₅₀/gram of tissue.

The replication of rHPIV2/V94 in the respiratory tract of hamsters wascompared to that of the biologically derived HPIV2/V94 to determinewhether the recovered rHPIV2/V94 retained the replicative properties ofits biological parent in vivo. In this regard, hamsters are accepted inthe art as a useful animal model of HPIV infection in humans thatprovides reasonably correlative findings for such activities asattenuation and immunogenicity between the model and humans, wherein themodel is understood to be a less permissive host than humans.

The two pools of the recombinant rHPIV2/V94, which were derived from twodifferent cDNA clones, were studied in parallel to assess thevariability in replication between two separate preparations of the samevirus. These were compared with the biologically derived preparation ofHPIV2/V94. Groups of 18 hamsters were separately inoculated intranasally(IN) with 10^(6.0) TCID₅₀ of each HPIV2. On days three, four, or five,the lungs and nasal turbinates were harvested from six hamsters fromeach group, and the level of replication of each virus was determined(FIG. 5). The level of replication of the two preparations of rHPIV2 wassimilar to that of the biologically derived HPIV2 on all of the daystested, demonstrating that the recombinant HPIV2/V94 had similarreplication properties in vivo as its biologically derived counterpart,and therefore, represents an authentic recombinant wt HPIV2/V94.Clearly, the incidental amino acid substitution at position 2093 of theL polymerase did not affect the level of replication of rHPIV2/V94 invivo. In addition to these studies, a full-length antigenomic cDNAencoding an alanine at position 2093 of the L protein, as occurs in thebiologically derived HPIV2/V94, was also generated, and this modifiedHPIV2/V94 cDNA which now would be identical in a sequence to theconsensus sequence for biologically derived HPIV2/V94, was also used torecover recombinant HPIV2/V94 in vitro.

EXAMPLE IV Determination of the Complete Genomic Sequences of ThreeStrains of HPIV2

Members of the Respirovirus and Morbillivirus genera of theParamyxovirinae subfamily of paramyxoviruses reportedly have beenconsidered to have a general requirement that the nucleotide length ofthe genome be an even multiple of six in order for efficient RNAreplication, and hence virus replication, to occur (Calain et al., J.Virol. 67:4822-30, 1993; Chanock et al. Parainfluenza Viruses. 4th ed.In “Fields Virology”, 4^(th) ed., Knipe et al., Eds., Vol. 1, pp.1341-1379, Lippincott Williams&Wilkins, Philadelphia, 2001; Hausmann etal., RNA 2:1033-45, 1996; Kolakofsky et al. J. Virol. 72:891-9, 1998;Peeters et al., Arch. Virol. 145(9), 1829-45, 2000, each incorporatedherein by reference). This “rule of six” was demonstrated in studieswith mini-replicons and is consistent with the observation that thecomplete genomic sequences for these viruses are even multiples of sixnucleotides. More recently, studies with parainfluenza virus type 3provided evidence that this “rule of six” operates at the level ofcomplete infectious virus (Skiadopoulos et al., Virology 297:136-152,2002). This rule is thought to be a consequence of each N proteinsubunit interacting with exactly six nucleotides in thevRNA-nucleoprotein complex.

However, it has remained unclear whether the rule of six applies to theRubulavirus genus of the paramyxoviruses. For example, studies withmini-replicons of simian virus type 5 suggested that there wasconsiderable flexibility with regard to this rule (Murphy et al.,Virology 232:145-57, 1997, incorporated herein by reference). Also, thecomplete genome sequence determined for the Toshiba strain of HPIV2 wasnot an even multiple of six, whereas that of HPIV2/V94 did conform tothe rule of six, and recombinant Toshiba strain HPIV2 was recovered fromcDNA-encoded antigenomic RNA that did not conform to the rule of six(Kawano et al., Virology 284:99-112, 2001, incorporated herein byreference).

Initial investigations herein focused on whether the natural genomelength of HPIV2 conformed to the rule of six. As noted above, thebiologically derived HPIV2 strain Vanderbilt 9412-6 (HPIV2/V94) wasobtained in 1994 from a 13 month old infant presenting with upper andlower respiratory tract infection, high fever and otitis media, andtherefore represents a recent, low passaged isolate of HPIV2. Thecomplete genomic sequence of HPIV2/V94 was determined from RT-PCRproducts amplified from purified vRNA. The sequence analysis wasperformed directly on RT-PCR products without a cloning step, and thusyields a consensus sequence. The HPIV2/V94 genome was found to be 15,654nt in length and thus conforms to the rule of six (Calain et al., J.Virol. 67:4822-30, 1993; Kolakofsky et al. J. Virol. 72:891-9, 1998,each incorporated herein by reference). Two additional HPIV2 strainswere also sequenced. The prototype HPIV2 Greer strain was originallyisolated in 1955 from an infected 11-month old infant (Chanock, J. Exp.Med. 104:555-76, 1956, incorporated herein by reference), and the HPIV2Vanderbilt 9811-18 (V98) strain was isolated in 1998 from an infected20-month old infant. The sequences for the V98 and Greer strains areprovided in FIGS. 10A-F and 11A-F, respectively, and by respectivereference GenBank accession no.s AF533011, AF533012 (each incorporatedherein by reference). The genome sizes for HPIV2/Greer and HPIV2/V98were both determined to be 15,654 nt, the same as HPIV2/V94. Thus, threestrains of HPIV2, isolated over 40 years apart, have identicalpolyhexameric genome lengths.

Sequence Comparison of the HPIV2 Strains

The nucleotide sequence of the Greer, V94 and V98 strains were comparedby the Gap sequence alignment program (Genetics Computer Group (GCG),Madison, Wis.). The percent identity among the three strains was 95.0%for the V94 and V98 strains; 97.4% for the V98 and Greer strains; and96.2% for the Greer and V94 strains. Thus, the recent V94 and V98isolates of HPIV2 are highly related to the 1955 Greer isolate. Thecis-acting transcription gene start (GS) and gene end (GE) signalsequences were highly conserved. Furthermore, the haxamer phasing of allknown cis-actin genomic sequences was exactly conserved among each ofthese viruses. Hexamer phasing refers to the position of any particularsequence motif within the respective genome hexamer(s). Correct hexamerphasing of cis-acting signals may be important for their optimalfunctioning (Kolalofsky et al. 1998) and is another ramification of therule of six. The predicted HPIV2/V94 N, P, V, M, F, HN and L polypeptidesequences were compared to Greer, V98 and several other members of theRubulaviruses (including SV5W3A strain, GenBank accession no. AF052755;SV41 strain ToshibalChanock, GenBank accession no. X64275; and mumpsvirus isolate 88-1961, GenBank accession no. AF467767, each incorporatedherein by reference) and the percent identity is shown in Table 1. Thepredicted polypeptide sequences are almost completely conserved betweenthe three HPIV2 strains, with the highest degree of divergence occurringin the HN glycoprotein.

TABLE 1 Amino acid sequence identity between the proteins of HPIV2/V94and the analogous proteins of HPIV2 Greer and V98 strains, SV41, SV5 andmumps virus Percent identity of HPIV2/V94^(a) with: HPIV2 Mumps ProteinGreer V98 SV41 SV5 virus N (543)^(b) 97.8 98.0 71.1 53.6 47.3 P (395)99.0 98.0 66.8 42.0 34.5 V (225) 99.6 97.3 68.9 42.6 32.2 M (377) 98.798.9 71.7 50.1 40.9 F (551) 99.1 98.0 59.6 45.9 37.4 HN (571) 96.1 94.960.8 45.8 39.0 L (2263) 99.7 99.2 77.8 62.1 59.0 ^(a)Percent identitybetween HPIV2/V94 and the analogous protein of the indicated virus wasdetermined by the Pileup program of the Wisconsin Package Version 10.2(Genetics Computer Group (GCG), Madison, Wisc.). ^(b)Number inparenthesis is the predicted amino acid length of the indicatedHPIV2/V94 protein.

The HPIV2 Toshiba strain (Genbank ascension number X57559; NC 003443,incorporated herein by reference) and the Greer strain were compared byBESTFIT sequence alignment (Wisconsin Package Version 10.2, GeneticsComputer Group (GCG), Madison, Wis., incorporated herein by reference)and were found to have 99.8% sequence identity. Thus, the Toshiba andGreer strains have a total of 34 nucleotide differences. Ten majorsequence discrepancies between the Toshiba and Greer strains wereidentified (FIG. 6). These included 13 nucleotides present in the Greerstrain that were missing from the reported Toshiba strain sequence, and5 additional nucleotides reported for the Toshiba sequence that were notpresent in the Greer strain. These included a 1-nt deletion in the N andP transcription gene start signal sequences, respectively, a 1-ntinsertion within the N gene end signal sequence, a 1-nt deletion in theHN gene 3′ NCR, and nt deletions or insertions in the N, P and L ORFsthat resulted in coding changes in those ORFs (FIG. 6). Correction forthese 18 errors in the Toshiba strain would yield a genome length of15654, the same size as reported here for the V94, V98 and Greer strainsof HPIV2.

Remarkably, nearly one half of the differences between the Toshiba andGreer strains involved apparent insertions or deletions. By far the mostcommon nucleotide differences between closely related paramyxovirusesinvolve nucleotide substitutions. Deletions and insertions are much morerare. The percentage of changes that involved cis-acting RNA signals orresulted in amino acid coding changes also was unusually high. The highfrequency of deletions and insertions in particular suggested that manyof the differences in the Toshiba strain represented errors in CDNAsynthesis, cloning or sequence analysis rather than true differences innature, and suggested that the observed non-compliance with the rule ofsix also might be due to error. This point, however, could not beresolved by sequence inspection alone.

EXAMPLE V Recombinant Mutant HPIV2s can be Generated from Full-lengthAntigenomic HPIV2 cDNAs that do not Conform to the Rule of Six

As noted above, the genome length of the Toshiba strain of HPIV2 wasreported to be 15646 nt (6n+4) (Genbank accession number X57559; NC003443) (Kawano et al., Virology 178:289-92, 1990a; Kawano et al.,Virology 179:857-61, 1990b; Kawano et al., Virology 174:308-13, 1990c;Kawano et al., Virology 284:99-112, 2001; Kawano et al., Nucleic AcidsRes. 19:2739-46, 1991, each incorporated herein by reference). Thisreported length is 4 nt longer than a length that would conform thegenome to be an even multiple of six and by convention its hexamerlength is designated 6n+4. In addition to these reports, an HPIV2Toshiba strain cDNA that did not conform to the rule of six (15665 nts;6n+5) was reportedly successfully used to recover a recombinant HPIV2 incell culture (Kawano et al., Virology 284:99-112, 2001). Based on thisinformation, it was previously thought that HPIV2, unlike other membersof the Paramyxovirinae (Chanock et al. Parainfluenza Viruses. 4^(th) ed.In “Fields Virology”, 4^(th) ed., Knipe et al., Eds., Vol. 1, pp.1341-1379, Lippincott Williams&Wilkins, Philadelphia, 2001) did not havea strict requirement for its genome length to conform to the rule of six(Kawano et al., Virology 284:99-112, 2001. Recently it was reported thatmutant recombinant HPIV3s can be derived from cDNAs that do not conformto the rule of six (Skiadopoulos et al., Virology 297:136-152, 2002),although HPIV3 has a requirement for a polyhexameric genome length, asdemonstrated with minigenome replication assays and sequencing of two wtHPIV3 genomes Durbin et al., Virology 235:323-332, 1997a; Durbin et al.,Virology 234:74-83, 1997b; Galinski, In “The Paramyxoviruses”, D. W.Kingsbury, Ed., pp. 537-568. Plenum Press, New York, 1991; Stokes etal., Virus Res. 25:91-103, 1992-published erratum appears in Virus Res.27:96, 1993, each incorporated herein by reference). Sequence analysisof HPIV3 recombinants derived from cDNAs that did not conform to therule of six indicated that they contained nucleotide insertions thatcorrected the length of the viral genome so that it conformed to therule of six (Skiadopoulos et al., Virology 297:136-152, 2002). Thisshowed that a) the complete infectious HPIV3 conforms to the rule ofsix, confirming the minigenomes studies, and b) that when HPIV3 isrecovered from antigenomic cDNA that does not conform to the rule ofsix, there apparently is an efficient mechanism that modifies bymutation the antigenome of genome length to bring it into strictcompliance with the rule of six.

The requirement for a polyhexameric genome length had not been directlyexamined for HPIV2, and it is not clear if HPIV2 requires apolyhexameric genome for efficient replication. To determine whetherHPIV2/V94 can be recovered from full-length cDNAs that do not conform tothe rule of six and to establish if these recovered viruses containgenome length correction mutations, full-length antigenomic cDNAs ofHPIV2/V94 were generated that were modified to each contain anoligonucleotide insert (FIG. 7) at an EcoRV restriction site near theend of the L polymerase gene. This site was chosen so as to notinterfere with the hexamer phasing of the upstream cis-acting gene start(GS) signal sequences, which may be important for efficient expressionof paramyxovirus mRNAs (Kawano et al., Virology 284:99-112, 2001, inch).Also, this region does not contain any known cis-acting sequences. Theinsertions served to maintain the L polymerase ORF sequence, butmodified the genome length such that the antigenomic HPIV2 cDNA lengthwas increased by 42 nt (6n with respect to hexamer spacing and conformsto the rule of six), 43 nt (6n+1), 44 nt (6n+2), 45 nt (6n+3), 46 nt(6n+4), or 47 nt (6n+5) (FIG. 7). These cDNAs were transfected intoHEp-2 cells along with HPIV2 support plasmids, as described above. Thesupernatants were harvested after 3 or 4 days and were passaged twice onLLC-MK2 monolayer cultures. Surprisingly, virus was readily recoveredfrom every cDNA tested, indicating that, although HPIV2 genomes arepolyhexameric, recombinant HPIV2 can be generated from a cDNA that doesnot conform to the rule of six.

Recombinant HPIV2s recovered from cDNAs that did not conform to the ruleof six (rHPIV2/V94 (+1), rHPIV2/V94 (+2), rHPIV2/V94 (+3), rHPIV2/V94(+4), and rHPIV2/V94 (+5)) were biologically cloned by plaquepurification on LLC-MK2 monolayer cultures and were further amplified bypassage on LLC-MK2 cells, as described above. rHPIV2/V94 (+1),rHPIV2/V94 (+2), rHPIV2/V94(+3), rHPIV2/V94(+4), and rHPIV2/V94(+5) werederived from cDNAs that were 15,697, 15,698, 15,699, 15,700 nts, or15,701 nts in length, respectively, which is 1, 2, 3, 4, or 5 more thanthat required to conform to the rule of six (6n+1, 6n+2, 6n+3, 6n+4 or6n+5, respectively). vRNA was isolated from each of these recoveredrecombinant viral isolates and was used to perform RT-PCR and RACE. ThePCR products were sequenced directly without cloning and a completeconsensus sequence of each viral genome was generated, as describedabove. This analysis showed that the genome length of viruses generatedfrom cDNAs that did not conform to the rule of six was modified in eachcase by nt insertions or deletions such that each recovered virusconformed to the rule of six (FIG. 8).

Complete genome sequencing of the rHPIV2/V94 (+3) vRNA RT-PCR productsrevealed that this virus had acquired a 2-nt (AA, antigenome sense)insertion in the HN gene 5′ non-coding region (NCR) and a 1-nt (T,antigenome sense) insertion within a poly-T tract in the HN gene 3′ NCR(FIG. 8A). The genome size of rHPIV2/V94 (+3) was 15,702 nts, i.e threents longer than the HPIV2 cDNA from which it was derived. Thus, the 3-ntinsertion served to modify the viral genome length so that it conformsto the rule of six. Complete sequencing of the rHPIV2/V94 (+4) genomealso showed that this virus had sustained a 2-nt insertion (AT,antigenome sense) at the end of the intergenic region between the HN andL genes and immediately upstream of the L gene GS signal sequence (FIG.8A). The genome size of rHPIV2/V94 (+4) was 15,702 nts, i.e two ntslonger than the 6n+4 HPIV2 cDNA from which it was derived. Thus, the2-nt insertion served to generate a polyhexameric genome length. Twoindependently derived, and biologically cloned rHPIV2s generated from afull-length antigenomic cDNA that was 15,701 nt (6n+5;r_(A)HPIV2/V94(+5) and r_(B)HPIV2/V94(+5)). r_(B)HPIV2/V94(+5) wascompletely sequenced and was found to have a single nt insertion in HN3′ untranslated region. The other (r_(A)HPIV2/V94(+5)) was partiallysequenced and was found to contain a single nucleotide insertion in theHN GE signal sequence that could serve to correct the genome length sothat it conforms to the rule of six (FIG. 8A). rHPIV2/V94(+1) andrHPIV2/V94(+2) were completely sequenced and were found to have 1 and 2nt deletions, respectively, that served to generate a polyhexamericgenome (FIG. 8B). The 2 nt deletion that was found in rHPIV2/V94(+2)occurred within the intergenic sequence between the HN and L genes.Interestingly, the single nt deletion in rHPIV2/V94(+1) occurred nearthe end of the coding region for the L polymerase and changed the 13amino acids encoded after codon 2250. Thus, the majority of genomelength corrections occurred at sites in non-coding regions but onemutant contained an alteration in the downstream end of an ORF.

In summary, the foregoing examples provide a detailed demonstration thatHPIV2 conforms to the rule of six, a demonstration that is particularlyrelevant because it was correlated with recovery of a complete,infectious recombinant virus. This discovery involved determination ofcomplete genomic sequences for three strains of HPIV2, namely theprototype Greer strain and two recent HPIV2 isolates, V94 and V98. Eachof these strains had a polyhexameric 15,654 nt genome length,demonstrating that the HPIV2 genome length is conserved and conforms tothe rule of six. A polyhexameric antigenomic HPIV2/V94 cDNA was used torecover a recombinant HPIV2/V94 and the length of the recovered viruswas also 15,654. Full-length antigenomic HPIV2 cDNAs that deviated fromthe rule of six by 1 to 5 nts yielded viruses that containednon-templated nt insertions or deletions which served to generatepolyhexameric genomes. These findings demonstrate that an HPIV2polyhexameric genome can be generated from a cDNA that does not conformto the rule of six, and that virus recovery in this context involvesspontaneous mutations that correct the length of the genome to conformto the rule of six. This demonstrates that HPIV2 requires apolyhexameric genome for successful recovery, and is consistent with thefindings that the genomes of the HPIV2 Greer, V98 and V94 strainsconform to the rule of six. Thus, the requirement for a polyhexamericgenome has now been demonstrated for members of the Rubulavirus andRespirovirus genera of the Paramyxovirinae subfamily, and suggests thatconformation to the rule of six and the ability to correct defects thatdeviate the genome length from the rule of six may be universal for allmembers of this paramyxovirus subfamily.

Comparison of the HPIV2 Toshiba strain and the Greer strain genomesequences reveals that these strains share 99.8% sequence identity. TheToshiba and Greer strains exhibit a total of 34 nucleotide differences.Several sequence discrepancies between the Toshiba and Greer strainswere noted. These included 13 nucleotides present in the Greer strainthat were missing from the reported Toshiba strain sequence, and 5additional nucleotides reported for the Toshiba sequence that were notpresent in the Greer strain. The high frequency of apparent deletionsand insertions in particular suggests that many of the differences inthe Toshiba strain represented errors in CDNA cloning, synthesis orsequence analysis—rather than true differences in nature, and alsosuggests that the previously reported non-compliance with the rule ofsix might be due to sequencing error. Correction for these 18 errors inthe Toshiba strain would yield a genome length of 15,654, the same sizeas reported here for the V94, V98 and Greer strains of HPIV2. Based onthese findings, it is likely that the recombinant Toshiba strain thatwas recovered previously from a cDNA that did not conform to the rule ofsix (Kawano et al., Virology 284:99-112, 2001, incorporated herein byreference) similarly sustained one or more nucleotide insertions or,potentially, deletions that would correct the genome to comply with therule of six.

The mechanism for the genome length correction observed here for HPIV2and previously for HPIV3 (Skiadopoulos et al., Virology 297:136-152,2002) derived from cDNAs that did not follow the rule of six is notknown, but two possibilities can be considered. First, nt insertions ordeletions may occur during the first step of the recovery of virus fromcDNA, during the (during the) synthesis of the initial antigenome RNAfrom the transfected antigenomic plasmid by the T7 RNA polymerase. Thebacteriophage T7 polymerase has been shown to insert or delete nts afterhomopolymeric A or T runs, at a rate of up to 1 in 1×4.5×10⁷ nt (Kroutilet al., Biochemistry 35:1046-1053, 1996, incorporated herein byreference).

The second possibility is that the nt modifications are generated by theHPIV2 polymerase. This could occur either by random ntinsertions/deletions that might arise during replication of the genomeor antigenome, or by a mechanism whereby the nucleoprotein replicationcomplex somehow detects a deviation in the genome length and adds ordeletes the appropriate number of nts to correct the defect and togenerate a polyhexameric length. Random insertion/deletionmisincorporations by either the T7 polymerase or the viral polymerasecomplex would be expected to occur at equal frequency at any positionthroughout the genome. Corrections occurring early within an openreading frame would likely be lethal for virus replication and generallyonly genomes with insertions in non-coding regions would be viable. Thefinding that the majority of the insertions or deletions reported herethat modified the length of rHPIV2/V94 occurred between the HN and Lgenes suggests that the genome length corrections did not occur by arandom insertion/deletion mechanism. Furthermore, viruses such asrHPIV2/V94 (+3) contained multiple insertion sites. Insertions atmultiple sites would be expected to occur at an even much lowerfrequency than single-site insertions, again suggesting that randominsertion is not the mechanism by which polyhexameric genome lengths aregenerated from cDNAs that do not conform to the rule of six.

Previously, we found that recombinant HPIV3 derived from cDNAs that didnot conform to the rule of six also contained nt insertions that servedto modify the genome length to conform to the rule of six. Theseinsertions (A or U) occurred either within a polyadenylation signalsequence (poly-U), or within a poly-A or poly-U tract of a foreign geneinsert contained within the HPIV3 genome. In the present study we alsodemonstrated a preference for homopolymeric A or U tracts as sites forinsertion, suggesting that the nt insertions may be the result ofpolymerase stuttering. The only exception was the AU insertion and an AUdeletion that occurred at an AUAU sequence in the IG region between theHN and L genes of rHPIV2/V94 (+4) and rHPIV2/V94 (+2), respectively,however these modifications may also be a result of polymerase slippageand stuttering. Interestingly, we have not observed any G or Cinsertions or deletions in any of the HPIV2 or HPIV3 constructs thathave been sequenced. It was previously shown that Sendai virusminigenomes whose lengths did not conform to the rule of six and whichcontained the P gene editing site (a poly-G stretch) underwent in vitront insertions or deletions within the editing site that served togenerate polyhexameric genome lengths (Hausmann et al., RNA 2: 1033-45,1996). DI genomes of SV5, a prototype virus of the Rubulavirus genus,were also shown to undergo in vitro genome length modification by ntinsertions, and SV5 DI genomes whose length conformed to the rule of sixreplicated more efficiently than uncorrected genomes (Murphy et al.,Virology 232:145-157, 1997, incorporated herein by reference). Othernegative-stranded RNA viruses have also been shown to generate modifiedgenomes that contain nt insertions. VSV, which is a member of theRhabdoviridae family and has a similar genomic organization asparamyxoviruses but does not follow the rule of six, has been shown toaccumulate many nt insertions in the 3′ NCR of the glycoprotein geneduring the natural evolution of the virus (Bilsel et al., J. Virol.64:4873-83, 1990, incorporated herein by reference). The nt insertionsidentified included homopolymeric stretches of U and/or As and it wassuggested that these occurred by polymerase stuttering. RSV, aparamyxovirus that does not follow the rule of six, is able to generateneutralization resistant mutants by inserting or deleting As within ahomopolymeric A stretch in the G protein gene (Garcia-Barreno et al., J.Virol. 68:5460-68, 1994; Garcia-Barreno et al., EMBO J. 9:4181-87,1990).

It is not clear from the present study if the ability to correctdeviations in genome length is an intrinsic property of the viralreplication machinery that is used during the natural evolution of thevirus to maintain proper genome length, or is an artifact of the reversegenetics system. The conservation of the length of the HPIV2 genome instrains isolated over 40 years apart, the numerous examples ofnegative-stranded RNA viruses that have the ability to modify theirgenome length, and the ease with which recombinant parainfluenza virusescan be derived from cDNAs that do not conform to the rule of sixsuggests that the former may be the case.

Summarizing the foregoing Examples, an authentic wild type (wt)rHPIV2/V94 was recovered from HEp-2 cells transfected with a full-lengthantigenomic HPIV2 cDNA and support plasmids expressing the HPIV2/V94 N,P and L proteins. The in vitro and in vivo growth characteristics of therecombinant HPIV2/V94 were generally indistinguishable to those of itsbiologically derived parent. Importantly, sequence analysis showed thatthe genome sequence of the recovered rHPIV2 was exactly consistent withthe antigenomic cDNA from which it was derived with the exception of asingle nt substitution. This substitution was not present in a secondisolate of recovered rHPIV2, indicating that it was an incidental pointmutation such as is commonly observed in RNA viruses. Thus apart fromthe occasional incidental mutations characteristic of RNA virus growth,it is possible to derive rHPIV3 in which the genome sequence is exactlyas designed. One requirement for success is conforming with the rule ofsix, which had previously been thought not to apply to HPIV2.

As discussed above, a reported HPIV2 strain (Toshiba) was previouslysequenced and its genome length (Genbank ascension number X57559) wasdisclosed to be 15,646 nt in length. Notably, this reported sequence is2 nt shorter or 4 Nt longer than the length shown here to be requiredfor conformation to the rule of six, and 8 nt shorter than the correctsequence reported here for HPIV2N94. Accordingly, this reported sequenceexceeds an even multiple of six by 4 nt (6n+4) (Kawano et al., Virology178:289-92, 1990a; Kawano et al., Virology 179:857-61, 1990b; Kawano etal., Virology 174:308-13, 1990c; Kawano et al., Virology 284:99-112,2001; Kawano et al., Nucleic Acids Res. 19:2739-46, 1991, eachincorporated herein by reference). In addition, an HPIV2 Toshiba straincDNA that did not conform to the rule of six was reported to yieldsuccessful recovery of a recombinant HPIV2 in cell culture (Kawano etal., 2001, supra). These observations provided strong support that HPIV2does not have a requirement for its genome length to conform to the ruleof six (Id.) On the basis of these reports, previous researchersconsidered that HPIV2 did not have a strict requirement for its genomelength to conform to the rule of six.

The rule of six was originally proposed for murine parainfluenza virustype I (MPIV1), also called Sendai virus, based on (1) the observationthat the nt length of the MPIV1 genome is an even multiple of six, and(2) studies with MPIV1 minireplicons, which showed that efficient RNAreplication was achieved only when the nt length of the minireplicon wasan even multiple of six (Calain et al. J. Virol. 67:4822-4831, 1993,incorporated herein by reference). Other reports propose that members ofthe Respirovirus and Morbillivirus genera of Paramyxoviridae generallyfollow the rule of six, based on the nt lengths of naturally occurringgenomes and on minigenome studies with a few representative viruses(Durbin et al., Virology 234:74-83, 1997b; Pelet et al., Virology224:405-141, 1996; Sidhu et al., Virology 208:800-71, 1995, eachincorporated herein by reference). This requirement for polyhexamericlength is thought to be a consequence of each N protein subunitinteracting with exactly six nucleotides in the vRNA-nucleoproteincomplex (Kolakofsky et al., J. Virol. 72:891-899, 1998; Vulliemoz etal., J. Virol. 75:4506-18, 2001, each incorporated herein by reference).It should be noted that the rule of six not been previously beenrigorously tested by systematic manipulation of complete infectiousvirus, which is the dispositive setting for determining the relevanceand significance of the rule to virus biology. Rather, availableexperimental evidence was based primarily on incomplete,helper-dependent, non-infectious minireplicons assayed in vitro.

The rule of six does not appear to apply to numerous other nonsegmentednegative strand RNA viruses, such as members of the Rhabdoviridae andFiloviridae families. The rule also does not apply to the Pneumovirusgenus of Paramyxoviridae (Samal et al., J. Virol. 70:5075-5082, 1996,incorporated herein by reference), represented by human RSV, andprobably also does not apply to the Metapneumovirus genus ofParamyxoviridae (van den Hoogen et al. Virology 295:119-132, 2002,incorporated herein by reference). Thus, most nonsegmented negativestrand viruses do not conform to the rule of six, and a similarrequirement that the genome nt length be a multiple of a specificinteger does not exist in any other type of virus.

The remaining genus of Paramyxoviridae, Rubulavirus, also did not appearto follow the rule of six. For example, minireplicon studies with SV5, aprototype Rubulavirus, indicated that a polyhexameric antigenome was notrequired for efficient RNA replication. (Murphy et al., Virology232:145-157, 1997, incorporated herein by reference).

Other previous observations were consistent with the idea thatRublaviruses, as exemplified by HPIV2, did not follow the rule of six.For example, in members of Respirovirus and Morbillivirus, which doconform to the rule of six, the positions of the first nt of the genestart (GS) signals of the various genes are largely conserved withregard to the hexameric phasing, suggesting that correct hexamer phasingof cis-acting signals is important for optimal functioning (Kolakofskyet al., 1998). Also, the members of these two genera have highlyconserved trinucleotide intergenic regions, whose precise and invariantlength also would be consistent with maintaining hexameric phasing ofcis-acting sequences. In contrast, the Rubulaviruses have a low degreeof conservation of the phasing of the GS signals (Kolakofsky et al., J.Virol. 72:891-899, 1998, incorporated herein by reference). Also,Rubulaviruses have highly variable intergenic regions: in the case ofHPIV2, these vary in length from 4 nt up to 45 nt. These findingssuggest that hexameric phasing of cis-acting signals is not importantfor HPIV2.

It has, therefore, become generally accepted in the field that the ruleof six was limited to two genera of Paramyxoviridae: Respirovirus andMorbillivirus. Also, as noted above, this rule was based largely on invitro studies with minireplicon systems, and its importance to completeinfectious virus and viral biology had not been clearly established. Inany case, this rule was thought to not apply to Rublaviruses. This wasview was strongly supported by the teachings of Kawano et al., 2001,supra, who disclosed the genome length of HPIV2 to be non-polyhexameric,and who further reported that a non-polyhexameric recombinantantigenomic cDNA produced infectious HPIV2. This report concerning HPIV2was particularly compelling, because the underlying studies involved thereported recovery of complete infectious virus, as opposed to simpleminireplicon RNA replication studies. The recovery of infectious virusfrom an antigenomic cDNA clone typically is relatively inefficient, suchthat a well of 1.5×10⁶ transfected cells might yield virus from 10 orfewer cells. Furthermore, any defect that reduced the replicationefficiency of infectious virus by even a small margin would quicklybecome apparent during multi-cycle virus growth. Hence, the reportedability to recover infectious virus was considered to provide a verystringent test that the cDNA-encoded antigenomic RNA was highlyfunctional. Therefore, the previous recovery of recombinant Toshibastrain HPIV2 from a non-polyhexameric antigenomic cDNA seeminglyprovided convincing evidence that the rule of six did not apply to thisvirus.

One of the key advantages of a cDNA based reverse genetics system forthe generation of viral variants is the ability to reproducibly recoverrecombinant viruses that contain a defined sequence. The goal ofproducing defined viral strains for development of useful immunogeniccompositions is possible only if an engineered antigenomic sequence canbe faithfully recovered in infectious virus. The reliable recovery ofdefined recombinant viruses is essential for studies to systematicallyadjust the level of viral attenuation and immunogenicity.

Unexpectedly, the present invention shows that the consensus sequencesdetermined for three additional strains of HPIV2, including two clinicalisolates of low passage history, conform to the rule of six. Comparisonwith the published Toshiba sequence provides evidence that the lattercontains numerous unpredictable nucleotide (nt) deletions andinsertions. While the origin and significance of theseinsertions/deletions are not known, such mutations are notcharacteristic of natural drift in this type of virus but rather arehallmarks of cloning or sequencing errors. Given the previous reportthat recombinant Toshiba strain HPIV2 was recovered from anon-polyhexameric antigenomic cDNA (Kawano et al., 2001, supra), it wasundertaken herein to systematically investigate the possibility ofrecovering infectious virus from a panel of antigenomic cDNAsspecifically engineered to span the full range of non-compliance withpolyhexamer phasing. Remarkably, recombinant virus was recovered readilyfrom each cDNA. These results would generally have been construed tovalidate the results reported by Kawano et al., as summarized above.However, detailed description and analysis of complete consensus genomicsequences presented herein demonstrate that each recovered virus did notcontain a faithful genomic copy of its respective antigenomic cDNA.Instead, each contained one or more nt insertions or deletions thatserved to confer a polyhexameric genomic length. This demonstrates thatthere is a rapid emergence of polyhexameric genomes in recombinant HPIV2viruses derived from non-polyhexameric antigenomes.

These findings have important implications for the development ofdefined mutant HPIV2 viruses and vectored immunogenic compositions byreverse genetics. Specifically, while virus can readily be recoveredfrom a HPIV2 cDNA that is not of polyhexameric length, the presentinvention shows that such virus will contain one or more unpredictableinsertions or deletions. The locations of these mutations cannot beclearly forecast, and the potential of these mutations to alter theproperties of the virus or vector with regard to growth, immunogenicity,expression of a supernumerary gene insert, pathogenicity, and otherproperties is equally unpredictable. Thus, the use of anon-polyhexameric antigenomic HPIV3 cDNA would generally be viewed tospecifically preclude the production of recombinant virus with a genomeof known sequence, since the recovered virus will have one or moreunpredictable insertions or deletions.

The discovery that natural isolates of HPIV2 have polyhexameric lengthgenomes, that HPIV2 strictly follows the rule of six at the level ofinfectious virus, and that non-polyhexameric antigenomic cDNAs readilygenerate mutated virus and must be avoided, was unanticipated andprovides the basis for the generation of recombinant HPIV2-basedimmunogenic compositions and viral vectors of defined sequence byreverse genetics.

In the present disclosure, recovery of virus from a HPIV2 cDNA that doesnot conform to the rule of six, and the demonstration that such virushas undergone length adjustments to conform to the rule of six,clarifies the requirement of HPIV2 to conform to the rule of six, andfurther evinces that the mechanism for this conformity isself-correction. In this context, previous understanding in the artconcerning HPIV2, which contemplated that this virus does not conform tothe rule of six, leads to a situation that would greatly complicate, andin the majority of cases preclude, the production of viruses of definedsequence. This is because a recovered virus that is not compliant withthe rule of six would be under a strong selective pressure toself-correct to conform to the rule of six, since this would confer moreefficient replication and allow the corrected virus to quickly outgrowthe starting virus. Such self-correction would involve nt insertions ordeletions to make the genome an even multiple of six. Indeed in theexamples in the present invention all viruses recovered from antigenomesthat did not comply to the rule of six contained mutations.

Furthermore, it is not possible to reliably predict where in the HPIV2genome such corrective changes might occur. For example, instances ofcorrection in the above-mentioned PIV3 system have been found bothwithin and outside of open reading frames, and within cis-actingsignals. Similarly, it is not possible to reliably predict what thephenotypic consequences of such changes might be. For example, there arenumerous instances of mutations that have minimal effect on growth invitro but are very deleterious on replication in vivo. This isespecially problematic for use of rHPIV2 as a vector, since asupernumerary foreign protective antigen, which is not required forreplication of the virus, would be a preferential target for the randominsertions/deletions that would serve to correct the genome. Theinsertion or deletion of nts would disrupt the reading frame orcis-acting regulatory sequences and potentially ablate expression of theforeign protective antigen. Thus, a recovery system that is notcompliant with the rule of six cannot reliably produce viruses ofdefined sequences and, indeed, would preferentially produce mutantviruses that have sustained length correction by Nt deletion orinsertion within the genome with potentially altered properties. Ittherefore would not be a desirable substrate for the generation oflive-attenuated candidate HPIV2 immunogenic compositions.

The recovery of virus from a cDNA not in compliance with the rule of sixhas the highly unpredictable effect of forcing mutations to occur. Aprincipal adverse consequence of this phenomenon is that one cannotreliably forecast where such length corrections might occur, nor is itpossible to cannot control the location(s) of such correction(s). Inaddition, such corrections almost always affect gene start phasing.Accordingly, the demonstration here of the dependence of HPIV2 on therule of six, and adherence to this rule by virus strains so recovered,is important for the production of virus of predetermined sequence.Conversely, the failure to recognize and adhere to this rule heretoforeprecluded recovery of virus having a defined, reproducible sequence, asopposed to a recombinant virus having mutations whose location, andphenotypic significance, could not have been clearly forecast.

The foregoing studies suggest that, when a virus is recovered from cDNAthat does not conform to the rule of six, two possible explanations canbe considered: (1) that the rule of six does not apply, or (2) lengthcorrection occurred. The second possibility previously seemed unlikely,because for paramyxoviruses insertions and deletions tend to be muchless frequent than nucleotide substitutions, and because it is difficultto imagine how such a length correction might occur with sufficientfrequency to account for the efficient recovery that was observed fornon-compliant cDNAs. Accordingly, previous reports rendered a strongconclusion that HPIV2 does not strictly obey the ‘rule of six’.

The present discoveries are therefore remarkable and unpredictable,because they show that length correction indeed does occur, andapparently occurs at an inexplicably high frequency. Previously, thefacile recovery of virus from non-compliant cDNAs was interpreted tomean that the rule of six does not apply strictly to HPIV2. From thisaccepted model, artisans would have proceeded to unknowingly developrecombinant viruses containing undisclosed mutations conferring unknownphenotypic properties. The present invention provides unexpectedinformation that will avoid this critical obstacle to the development ofviruses and vectors from recombinant HPIV2.

EXAMPLE VI Importation of Mutations Identified in HeterologousParomyxoviruses into the L Protein of HPIV2

Within other aspects of the present invention, various attenuatingmutations identified in heterologous paramyxoviruses can be introducedinto a recombinant HPIV2, singly or in combination, to obtain a suitabledegree of attenuation or other desired phenotypic effects. For example,specific mutations that confer the attenuation phenotype of HPIV3 cp45can be introduced at a corresponding target site in rHPIV2. Additionalattenuating mutations have been developed by “importing” attenuatingpoint mutations from RSV and BPIV3 into a recombinant HPIV. In certainembodiments, point mutations are introduced into recombinant viruses ofthe invention using two or three nucleotide changes at a codonspecifying the mutation, rather than one, which will stabilize themutation against reversion to wild type.

Since attenuating mutations have not been previously described oravailable for HPIV2, the use of heterologous mutations is particularlydesired to attenuate rHPIV2 of the invention. Using sequence alignmentsas a guide, several mutations identified in the L protein of HPIV3 cp45,and in a novel attenuated chimeric bovine-human recombinant designatedrHPIV3-L_(B), were used as exemplary mutations to producelive-attenuated HPIV2 for use in immunogenic compositions. Theseincluded recombinants bearing mutations at positions 460, 948, 1565, andamino acids 1724-1725 (Δ1724) of the L polymerase. Recombinantsrecovered from HPIV2 cDNA were biologically cloned and were confirmed tocontain the appropriate mutation specified by the cDNA using RT-PCR ofpurified vRNA as described previously. Each of the four mutantrecombinants bearing HPIV2 L mutations grew to high titer (≧7.8 log₁₀TCID₅₀/ml) indicating that replication was not restricted at permissivetemperature (32° C.). Unexpectedly, one mutant, rHPIV2: Δ1724,containing a 2 amino acid deletion in the L protein (note that twocodons were deleted in this mutant to conform to the rule of six,although the corresponding target site of interest for importing themutation is residue 1724) grew to almost 10⁹ TCID₅₀/ml—indicating thatits growth in vitro was also unaffected by the mutation in the Lprotein. These exemplary mutations and other mutations identified inheterologous paramyxoviruse can be introduced into a rHPIV having othernucleotide modifications, for example chimeric and vector HPIV2constructs as described herein.

Replication of Mutant rHPIV2s in LLC-MK2 Cells at Permissive andRestrictive Temperatures

The ts phenotype for each of the four exemplary mutant rHPIV2 viruseswas determined by comparing its level of replication to that of rHPIV2wild type at 32° C. and 38° C. and 39° C. as described previously(Skiadopoulos et al., Vaccine 18:503-510, 1999, incorporated herein byreference). Briefly, each virus was serially diluted 10-fold in 96-wellLLC-MK2 monolayer cultures in L-15 media (Quality Biologicals orGibco-Invitrogen, Inc.) and antibiotics. Replicate plates were incubatedat the temperatures indicated above for six days, and virus infectedcultures were detected by hemadsorption with guinea pig erythrocytes.Virus titer at each temperature was determined in two to six separateexperiments and is expressed as the log₁₀ 50 percent tissue cultureinfectious doses per milliliter (TCID50/ml). The reduction in titer atelevated temperature was compared to the titer at 32° C., and a meanreduction in titer was determined. The shut-off temperature of a rHPIV2mutant is defined as the lowest temperature at which the reduction invirus titer compared to its titer at 32° C. was 100-fold greater thanthat of wild type rHPIV2 at the same temperature. A mutant is defined astemperature sensitive if its reduction in replication at 39° C., i.e.,the titer at 32° C. minus that at 39° C., is 100-fold or greater thanthat of wild type rHPIV2.

As a reference in some experiments, a recombinant HPIV1 L proteinmutant, rHPIV1 L: Y942Hcp45, was concurrently tested as described aboveexcept trypsin was added to the growth medium. The four exemplary rHPIV2viruses bearing mutations imported from RSV, HPIV3 or rHPIV3-L_(B) weretested for their ability to replicate at permissive temperature (32° C.)versus a range of higher temperatures (38-39° C.) by titration onLLC-MK2 monolayers (Table 2). Interestingly none of the ts and attmutations imported from RSV or HPIV3 cp45 specified a ts phenotype inrHPIV2. However, the mutation targeting residue 1724 of the L proteinspecified a ts phenotype with a shutoff temperature of 39° C.

TABLE 2 Replication of biologically derived and recombinant HPIV2 atpermissive and restrictive temperatures Titer^(a) at indicatedtemperature (° C.) (log₁₀ TCID₅₀/ml) Virus 32 38 39 rHPIV2/V94 (Not) wt8.1 8.5 7.3 rHPIV1 L: Y942H_(cp45) 8.1 2.6 2.5 rHPIV2 L:F450L 8.6 8.07.7 rHPIV2 L:Y948H 8.6 8.2 7.1 rHPIV2 L:L1565I 8.0 7.7 7.1 rHPIV2L:Δ1724 7.8 6.3 4.5 ^(a)Values in bold type are at or below the shut-offtemperature, which is defined as a 100-fold or more reduction in titercompared to the titer at 32° C. while correcting for the loss of wildtype titer.

Replication of Biologically Derived and rHPIV2 Mutant Viruses in theRespiratory Tract of Hamsters

The ability of these exemplary mutant HPIV2 strains to replicate in therespiratory tract of hamsters was evaluated as follows. Four to fiveweek-old Golden Syrian hamsters were inoculated intranasally with 0.1 mlof L-15 medium containing 10^(6.0) TCID₅₀ of a wild type or mutantHPIV2. Lungs and nasal turbinates were harvested on day fourpost-infection, and the titer of virus was determined as previouslydescribed (Skiadopoulos et al., Vaccine 18:503-510, 1999). The meanlog₁₀ TCID₅₀/g was calculated for each group of six hamsters. Each ofthe wild type, biologically derived strains of HPIV2 replicated to hightiter in the upper and lower respiratory tract of hamsters. The level ofreplication of the recombinant HPIV2 was similar to that of itsbiologically derived parent virus. The ability of the four rHPIV2mutants containing a mutation imported from RSV, HPIV3 cp45, orrHPIV3-LB to replicate in the upper and lower respiratory tract ofhamsters was examined. The level of replication of the rHPIV2 mutants inthe upper (nasal turbinates) and lower (lungs) respiratory tract ofinfected hamsters was compared to that of wild type recombinant HPIV2(Table 3). Among the single amino acid mutations, recombinant HPIV2bearing the F460L or L1565I mutations in the L protein exhibited a100-fold or greater reduction in replication in the lower respiratorytract of hamsters compared to the recombinant rHPIV2/V94 (Not) parentvirus. These results demonstrate that the two subject mutations specifyan in vivo attenuation phenotype for rHPIV2.

TABLE 3 Replication of biologically derived strains of HPIV2 andrecombinant wild type or mutant HPIV1/V94 in the respiratory tract ofhamsters Mean virus titer No. of (log₁₀ TCID₅₀/g ± S.E.) Virus^(a)animals Nasal Turbinates Lungs HPIV2/Greer wild 4 6.1 ± 0.1 5.8 ± 0.1type HPIV2/V98 wild type 6 5.4 ± 0.2 4.8 ± 0.4 HPIV2/V94 wild type 6 4.9± 0.2 5.2 ± 0.8 rHPIV2/V94 (Not) 6 5.2 ± 0.1 5.5 ± 0.3 rHPIV2 L:F460L 65.0 ± 0.1 3.1 ± 0.3 rHPIV2 L:Y948H 6 5.6 ± 0.1 4.5 ± 0.4 rHPIV2 L:L1565I6 4.6 ± 0.4 3.1 ± 0.5 rHPIV2-F_(RSV) 6 4.1 ± 0.3 2.1 ± 0.4 ^(a)Hamstersin groups of 6 or 4 were inoculated IN with 10⁶ TCID₅₀ of the indicatedvirus. Nasal turbinates and lung tissues were harvested on day 4post-infection, and virus present in the tissues was quantified byserial dilution on LLC-MK2 monolayers incubated at 32° C. The mean virustiter ± standard error (S.E.) is shown. Values in bold show a 100-foldor more reduction in titer compared to the titer of rHPIV2/V94.

Attenuation of rHPIV2 in Non-Human Primates

The replication of the rHPIV2-L: Δ1724 recombinant was compared to thatof the wild type HPIV2/V94 in an accepted non-human primate model forevaluating attenuation of recombinant HPIVs for use in immunogeniccompositions in humans (African green monkeys seronegative for HPIV2).The monkeys were inoculated intranasally (IN) and intratracheally (IT)with one milliliter of L15 medium containing 10⁶ TCID₅₀ of virussuspension, as described previously (Durbin et al., Vaccine 16:1324-30,1998). Nasopharyngeal swab samples were collected on days 1 through 10post immunization, and tracheal lavage samples were collected on days 2,4, 6, 8, and 10 post-immunization. Virus present in the collectedsamples was quantified by serial dilution on LLC-MK2 monolayer culturesat 32° C. and is expressed as log₁₀TCID₅₀/ml (Table 4). The biologicallyderived virus replicated to a high level in the lower respiratory tractand to a lower level in the upper respiratory tract (FIG. 12). TherHPIV2-L:Δ1764 mutant was attenuated for replication in both the upperand lower respiratory tracts relative to its biologically derived parentvirus. This identified the L:Δ1724 mutation as one that confers a highlevel of attenuation to rHPIV2 in primates. Because this mutation is atwo amino acid deletion, the subject recombinant virus will be highlystable following replication in vivo-a highly desired characteristic fora candidate virus for use in immunogenic compositions and methods.

TABLE 4 Level of virus replication of biologically derived andrecombinant HPIV2 in African green monkeys Mean peak virus titer^(b) ±S.E. (log₁₀TCID₅₀/ml) in: Virus administered^(a) NP swab TL HPIV2/Greerwild type 2.7 ± 0.6 3.7 ± 0.5 HPIV2/V98 wild type 2.6 ± 0.6 4.6 ± 0.5HPIV2/V94 wild type 2.5 ± 0.6 5.2 ± 0.3 rHPIV2/V94 (Not) wt 1.9 ± 0.53.4 ± 0.2 rHPIV2 L: Δ1724 1.3 ± 0.5 2.0 ± 0.2 ^(a)Animals were infectedIN and IT with 10⁶ TCID₅₀ of the indicated virus. ^(b)Nasopharyngeal(NP) swab samples were collected on days 1 to 10 post-infection.Tracheal lavage (TL) samples were collected on days 2, 4, 6, 8 and 10post-infection. Mean of the peak virus titer for each animal in itsgroup irrespective of sampling day. S.E. = standard error. The lowerlimit of detection of virus titer is 10 TCID₅₀/ml.

EXAMPLE VII Use of the Wild Type rHPIV2/V94 as a Vector to ExpressProtective Antigens of Heterologous Pathogens

In other aspects of the invention, compositions and methods are providedthat employ various recombinant HPIV2 constructs as vectors to expressone or more protective antigen(s) of a heterologous pathogen, forexample as a substitute or supernumerary gene or genome segment.Heterologous pathogens of interest in these aspects of the inventioninclude, for example, heterologous PIVs, measles virus, humanmetapneumovirus (HMPV), and RSV.

As noted above, HPIV1, 2 and 3 represent different serotypes that do notnaturally provide cross protection, and hence effective immunogeniccompositions must be designed specifically against each serotype virus.In addition, recombinant HPIV2 viruses of the invention have propertiesthat make them particularly useful as vectors to express the protectiveantigens of other microbial pathogens, including other HPIVs.

The major protective antigens of HPIVs and of certain othermononegavirus pathogens, such as measles virus and respiratory syncytialvirus (RSV), are the two major surface proteins that mediate attachmentand penetration of the host cell. These are the HN and F glycoproteinsof the PIVs, the HA and F glycoproteins of measles virus, and the G andF proteins of RSV. Importantly, for each of these viruses, eitherglycoprotein expressed alone functions as a neutralization andprotective antigen. In contrast, “internal” proteins of these virusescan induce protective immunity that appears to be mediated by CD8⁺cytotoxic T cells, but this protective effect wanes within a matter ofmonths and does not appear to be a significant component of long livedresistance to reinfection (Connors et al., J Virol 65:1634-71991;Kulkami et al., J Virol 67:1044-9, 1993; Tao et al., Vaccine17:1100-1108, 1999). Numerous studies have shown that expression of evena single mononegavirus protective glycoprotein antigen can elicit a highlevel immune response in mammalian subjects, which may include bothlocal and systemic immunity-despite neutralizing and immunosuppressiveeffects of maternal antibodies present in infants and young children.Unexpectedly, HPIV2 vectors of the invention are particularly wellsuited as vectors for immunization against a number of pathogens,especially for pediatric populations.

While several members of the Paramyxovirus family have been shown to besuitable as expression vectors, a recombinant HPIV2 expression vectorhas not been previously described. Modification of a single recombinantvirus to induce immunity against multiple pathogens has severaladvantages. It is more feasible and expeditious to develop a singleattenuated chimeric viral “backbone” construct expressing antigens thatelicit a polyspecific immune response against multiple pathogens, thanit is to develop separate attenuated viruses for each pathogen. Eachpathogen offers different challenges for manipulation, attenuation anddemonstration of safety and efficacy, and it would be a daunting task todevelop an attenuated version of each of a series of pathogens. It isconsiderably more efficient to develop, prepare, handle, and administera single immunogenic virus than it is to administer several attenuatedviruses. Reducing the number of immunizing viruses also will simplifythe crowded schedule of pediatric immunizations. Several attenuatedviruses can be administered as a mixture, but this complicatesdevelopment of clinically applicable immunogenic compositions, sinceeach component must be shown to be safe separately, and then shown to besafe and efficacious as a mixture. One particular problem with theadministration of mixtures of viruses is the common phenomenon of viralinterference, in which one or more of the viruses in the mixtureinterferes with the replication of one or more of the other components.This may result in reduced replication and immunogenicity for one ormore components, which is obviated by the use of a single vectorbackbone. Also, since some viruses such as measles virus have particularsafety concerns, as described below, it is safer to use a singlecomparatively benign virus such as HPIV2 as a vector bearing multiplesupernumerary antigens, as opposed to a mixture of separately-attenuatedviruses, each of which must be developed and validated separately.

The HPIV2 vector system has unique advantages over other members of thesingle-stranded, negative-sense viruses of the Order Mononegavirales.First, most other mononegaviruses that have been used as vectors are notderived from human pathogens e.g. murine HPIV1 (Sendai virus) (Sakai etal., FEBS Lett 456: 221-61999), vesicular stomatitis virus (VSV) whichis a bovine pathogen (Roberts et al., 1998), and canine PIV2 (SV5) (Heet al., Virology 237:249-60, 1997). For these nonhuman viruses, littleor only weak immunity is conferred against any human virus by antigenspresent in the vector backbone. Thus, a nonhuman virus vector expressinga supernumerary gene for a human pathogen will generally only elicit aneffective immune response against that single human pathogen. Inaddition, use of non-human viruses such as VSV, SV5, rabies, or Sendaivirus as vector requires exposure of subjects to viruses that theylikely would not otherwise encounter during life. Infection with, andimmune responses against, such nonhuman viruses poses additional safetyconcerns because there is little experience of infection with theseviruses in humans.

Three human mononegaviruses that have been proposed for use as vectors,measles, mumps, and rabies viruses, have additional limitations thatmake them poor candidates for this purpose. For example, measles virushas been considered for use as a vector for the protective antigen ofhepatitis B virus (Singh et al., J Virol 73: 4823-8, 1999). However,this combined measles virus-hepatitis B virus vaccine could only begiven, like the licensed measles virus vaccine, after nine months ofage, whereas the current hepatitis B virus vaccine is recommended foruse in early infancy. This is because the currently licensed measlesvirus vaccine is administered parenterally and is very sensitive toneutralization and immunosuppression by maternal antibodies, andtherefore is not effective if administered before 9-15 months of age.Thus, it could not be used to vector antigens that cause disease inearly infancy and therefore would not be useful for eliciting effectiveimmune responses in these subjects against RSV and HPIVs. Another wellknown, characteristic effect of measles virus infection isvirus-mediated immunosuppression, which can last several months and isnot a desirable feature for a vector. The attenuated measles virusvaccine was associated with altered immune responses and excessmortality when administered at increased dose, which might be due atleast in part to virus-induced immunosuppression and indicates that evenan attenuated measles virus might not be appropriate as a vector.Furthermore, the use of measles virus as a vector would be inconsistentwith the global effort to eradicate this pathogen. Indeed, for thesereasons it would be desirable to end the use of live measles virus andreplace the present measles virus vaccine with a PIV vector thatexpresses measles virus protective antigens, as described herein.

Rabies virus, a rare cause of infection of humans, has been alsoconsidered for use as a vector (Mebatsion et al., Proc. Natl. Acad. Sci.U.S.A. 93:7310-4, 1996), but it is unlikely that a vector that is 100%fatal for humans would be developed for use as a live attenuated virusvector, especially since immunity to the rabies virus, which is not aubiquitous human pathogen, is not needed for the general population.While mumps and measles viruses are less pathogenic, infection by eithervirus can involve undesirable features. Mumps virus infects the parotidgland and can spread to the testes, sometimes resulting in sterility.Measles virus establishes a viremia, and the widespread nature of itsinfection is exemplified by the associated widespread rash. Mildencephalitis during mumps and measles infection is not uncommon. Measlesvirus also is associated with a rare progressive fatal neurologicaldisease called subacute sclerosing encephalitis.

In contrast, PIV infection and disease in normal individuals is limitedto the respiratory tract, a site that is much more advantageous forimmunization than the parental route. Viremia and spread to secondarysites can occur in severely immunocompromised experimental animals andhumans, but this is not a characteristic of typical PIV infection. Acuterespiratory tract disease is the only disease associated with PIVs.Thus, use of PIVs as vectors will, on the basis of their biologicalcharacteristics, avoid complications such as interaction of virus withperipheral lymphocytes, leading to immunosuppression, or infection ofsecondary organs such as the testes or central nervous system, leadingto other complications.

An important and specific feature of PIV vector systems is that they maybe administered intranasally, mimicking natural infection and inducingboth mucosal and systemic immune responses. PIV vector systems will alsoreduce neutralizing and immunosuppressive effects of maternally-derivedserum IgG that is present in infants. In addition to these advantages,conditions have been established to obtain high titers of PIV3 inmicrocarrier culture that are 10 to 1000 times greater than can beachieved with viruses such as RSV and measles virus. Additionaladvantages of a PIV vector system relate to ease of propagation,transport, storage and handling compared to other contemplated vectorsystems.

The recombinant HPIV2 viruses described here can be used as vectors forexpressing the protective antigens of heterologous human pathogens, forexample by inserting a supernumerary gene encoding one or more antigenicdeterminants of a different pathogen, such as another Paramyxovirus. Forexample, the F and/or HN protein(s) of HPIV1 or HPIV3, or the F and/or Gglycoprotein(s) of respiratory syncytial virus (RSV) (or one or moreantigenic domains, segments, or epitopes thereof) can be substituted orinserted as a supernumerary gene or genome segment into a recombinantHPIV2 backbone or vector. Exemplary design of such a chimeric rHPIV2 isillustrated in FIG. 2. In this example, a unique restriction enzymeendonuclease recognition sequence (Not I) is introduced by PCRmutagenesis immediately upstream of the translation initiation codon(AUG) of the HPIV2 N gene (nts 158-160). This can then be used to insertan operable coding sequence corresponding to an antigen or antigenicdeterminant of a heterologous virus. In exemplary embodiments an openreading frame (ORF) encoding a heterologous antigen is placed underHPIV2 cis-acting transcription control elements and is transcribed as anadditional mRNA.

Use of Recombinant HPIV2 as an Expression Vector for SupernumeraryForeign Genes Including the RSV F Glycoprotein and Green FluorescentProtein (GFP).

Thus, in one exemplary aspect of the invention, the superiorcharacteristics of rHPIV2 vectors provide for development of immunogeniccompositions effective to elicit an immune response against RSV. In thepresent example, HPIV2 cDNAs were constructed containing gene unitsexpressing the RSV subtype A fusion protein (F) ORF or a greenfluorescent protein (GFP) ORF inserted upstream of the N coding sequenceand under the control of the cis-acting HPIV2 transcription signals.From these cDNA constructs, recombinant HPIV2 vector viruses expressingthe RSV F protein or GFP were recovered. Recombinants were biologicallycloned and high titer pools were generated (≧7.0 log₁₀ TCID₅₀/ml).Expression of the GFP was confirmed by fluorescent microscopy,indicating that supernumerary genes inserted upstream of the HPIV2 Ngene are well tolerated. Unexpectedly, the recombinant containing anadditional gene encoding the RSV F protein (rHPIV2-FRSV) was moderatelyattenuated for replication in vitro (FIG. 13). This virus grew to10^(7.6) TCID₅₀ in cell culture. However, its rate of growth was reducedcompared to wild type HPIV2. Expression of the RSV fusion protein wasconfirmed by indirect immunofluorescence using monoclonal antibodies tothe RSV fusion protein as described previously (Skiadopoulos et al., JVirol 70:1117-24, 1996; Skiadopoulos et al., Virology 297: 136-52, 2002;Schmidt et al., J Virol 75: 4594-603, 2001)). Thus this recombinantexpresses the protective antigens of two human pathogens, RSV and HPIV2.

The level of replication of rHPIV2-FRSV in hamsters was determined byinoculating groups of 6 hamsters IN with 10⁶ TCID₅₀ of rHPIV2-FRSV orrHPIV2 wt and determining the level of replication of each virus in thelungs and nasal turbinates on day 4 post-infection. As shown in Table 3,the rHPIV2-FRSV was 10-fold reduced for replication in the upperrespiratory tract, and 2500-fold reduced for replication in the lowerrespiratory tract. This level of attenuation was unexpected becausesimilar chimeric bovine-human PIV3 and HPIV1 recombinants bearing theRSV F gene in the first transcriptional position were not attenuated invitro or in vivo. This identifies rHPIV2-FRSV as a novel class of HPIV2mutant; an HPIV2 expression vector attenuated by virtue of a single geneunit insertion upstream of the N gene.

EXAMPLE VIII Additional Analyses of Attenuating Mutations in rHPIV1 forDevelopment of Immunogenic Compositions and Methods

As noted above, the instant invention also provides novel recombinantHPIV1 viruses and related compositions and methods that are useful aloneand/or in combination with the HPIV2 compositions and methods describedherein. For economy of description, foundational aspects of theseembodiments are described, for example, in U.S. patent application Ser.No. 10/302,547, filed by Murphy et al. on Nov. 21, 2002 and in thecorresponding PCT Publication Number WO 03/043587 A2, published on May30, 2003 (each incorporated herein by reference). Mutations and othermodifications identified and characterized for rHPIV1 viruses of theinvention can be readily incorporated and evaluated in rHPIV2 virusesdescribed herein, and vice versa.

In exemplary embodiments, the recombinant HPIV1 genome or antigenomeincorporates one or more recombinantly-introduced, temperature sensitive(ts) or host range (hr) attenuating (att) mutations. Often, therecombinant HPIV1 genome or antigenome will incorporate one or moreattenuating mutation(s) identified in a biologically derived mutant PIVstrain, or in another mutant nonsegmented negative stranded RNA virus,for example RSV or murine PIV (MPIV). For example, the recombinant HPIV1genome or antigenome can be modified or constructed to incorporate oneor more mutation(s) corresponding to mutation(s) identified in a HPIV1,or a heterologous PIV such as the well known immunogenic compositioncandidate HPIV3 JS cp45. Useful mutations of HPIV3 JS cp45 or anothermutant virus can specify a change in a HPIV1 protein selected from L, M,N, C, F, or HN or in a HPIV1 extragenic sequence selected from a 3′leader or N gene start sequence. Where the mutation relates to aparticular amino acid residue, the recombinant HPIV1 genome orantigenome will often incorporate multiple nucleotide changes in a codonspecifying the mutation to stabilize the modification against reversion.

The large polymerase protein (L) is highly conserved at the amino acidlevel between human parainfluenza virus type 1 and type 3 (HPIV1 andHPIV3). The Y942H and L992F temperature sensitive (ts) and attenuatingamino acid substitution mutations, previously identified in the Lpolymerase of the HPIV3-cp45 vaccine candidate, were introduced intohomologous positions of the L polymerase of recombinant HPIV1 (rHPIV1).In rHPIV1, the Y942H mutation specified the ts phenotype in vitro andthe attenuation (att) phenotype in hamsters, whereas the L992F mutationspecified neither phenotype. Each of these two mutations in bothHPIV3-cp45 and rHPIV1 was generated by a single nucleotide substitutionand, therefore, had the potential to readily revert to a codonspecifying the wild type amino acid residue. In the present Example, theeffects of introducing alternative amino acid assignments at eithercodon was evaluated as a strategy to increase genetic stability and toexplore the range of possible attenuation phenotypes for rHPIVs of theinvention.

An HPIV1 antigenomic cDNA was molecularly engineered to specify each ofthe other 18 amino acids at position 942 of the L protein or 17 of the18 other amino acids at position 992. Thirteen rHPIV1 codon substitutionmutants with alternative amino acid substitutions at position 942 and 10mutants at position 992 were shown to be viable. At position 942, anumber of mutants with a similar level of temperature sensitivity andattenuation as the Y942H virus were identified, several of whichdiffered by three nucleotides from either of the two codons encoding thewild type assignment of Tyr. One such mutant, the Y942A virus, wasdirectly confirmed to possess a high level of genetic and phenotypicstability upon serial passage in vitro at successively elevatedrestrictive temperatures compared to the Y942H virus involving a singlenucleotide substitution, which was relatively unstable. At position 992,three substitution mutants, L992V, L992C and L992N, were obtained that,in contrast to the L992F virus, possessed the ts and att phenotypes.These findings identify codon substitution mutations that specifyincreased genetic stability and/or increased attenuation, propertiesthat are highly desirable for mutations in a live attenuated HPIV1 orHPIV2.

In the HPIV3 backbone, the Y942H and L992F mutations each specify the tsphenotype in vitro and the att phenotype in hamsters. One objective inthe present Example was to determine the effect of each mutation in theHPIV1 backbone, and to validate a general strategy for increase thegenetic and phenotypic stability of ts and att amino acid pointmutations in live attenuated HPIVs. The high mutation rate for RNAviruses in general renders single-nucleotide changes susceptible togenetic and phenotypic instability. Since one mechanism of loss of thets phenotype is reversion of the nucleotide substitution to the wildtype assignment, we sought to modify the rHPIV1 codons such that two orthree nucleotide changes would be required to restore the wild typeamino acid coding assignment. This strategy has been previously employedto generate a set of attenuated recombinant Sindbis viruses bearingcodon substitution mutations at several sites in the E2 virion protein.(Polo et al., J Virol 65:6358-61, 1991; Schoepp et al., Virology193:149-59, 1993). These recombinant viruses exhibited different levelsof attenuation in vivo, but their phenotypic stability in vitro or invivo was not explored. Thus, we used mutagenesis at rHPIV1 codons 942and 992 to explore the range of viable amino acid coding assignments ateach position and recovered 13 rHPIV1 viruses each with a differentamino acid at position: 942 and 10 viruses each with a substitution atposition 992. These codon substitution mutants were assayed fortemperature sensitivity of replication in vitro and for their ability toreplicate in the respiratory tract of hamsters. rHPIV1 viruses withincreased attenuation or genetic stability were identified in bothcodons. These mutations will be useful as attenuating mutations inrHPIV1 and rHPIV2 for development of effective immunogenic compositionsand methods.

Viruses and Cells

LLC-MK2 cells (ATCC CCL 7.1) and HEp-2 cells (ATCC CCL 23) weremaintained in Opti-MEM I (Gibco-Invitrogen, Inc. Grand Island, N.Y.)supplemented with 5% FBS, gentamicin sulfate (50 μg/ml), and 2 mMglutamine (Gibco-Invitrogen, Inc.) The recombinant HPIV1 (rHPIV1) andrHPIV1 mutants were grown in LLC-MK2 cells as described previously.(Newman et al., Virus Genes 24:77-92, 2002).

Construction of point mutations in the antigenomic HPIV1 cDNA. Themutations were introduced into the appropriate rHPIV1 subgenomic clones(Newman et al., Virus Genes 24:77-92, 2002) using a modified PCRmutagenesis protocol described elsewhere (Moeller et al., J Virol75:7612-20, 2001) with the Advantage-HF PCR Kit (Clontech Laboratories,Palo Alto, Calif.). The subgenomic clone containing the mutation wasthen sequenced for the entirety of the region that was PCR-amplifiedusing a Perkin-Elmer ABI 3100 sequencer with the BigDye sequencing kit(Perkin-Elmer Applied Biosystems, Warrington, UK) to confirm that thesubclone contained the introduced mutation but did not contain anyadventitious mutations introduced during PCR amplification. Full-lengthHPIV1 cDNA clones (FLCs) containing the mutations were assembled usingstandard molecular cloning techniques (Newman et al., Virus Genes24:77-92, 2002), and the region containing the introduced mutation ineach FLC was sequenced as described above to ensure that the FLCcontained the introduced mutation.

Recovery of rHPIV1 Mutant Viruses

Recovery of rHPIV1 mutants was performed as described previously (Newmanet al., Virus Genes 24:77-92, 2002). To confirm that viruses containedthe appropriate mutations, viral RNA (vRNA) was isolated from infectedcell supernatant fluids using the Qiaquick vRNA kit (Qiagen Inc.,Valencia Calif.), and the appropriate region in each was amplified byRT-PCR as described previously (Newman et al., Virus Genes 24:77-92,2002) and analyzed by sequencing. All of the sequence analysis of viralRNA in this study involved direct analysis of uncloned RT-PCR products.Control RT-PCR reactions were performed in which the RT enzyme wasomitted to confirm that the RT-PCR products were generated from RNArather than contaminating DNA. For FLCs containing a codon substitutionmutation, initial virus recovery attempts were made as described aboveusing the pTM(L1) support plasmid that contained the HPIV1 wild type Lprotein sequence (Newman et al., Virus Genes 24:77-92, 2002). In severalinstances involving the 942 or 992 codon the virus recovered containedthe wild type L coding protein coding sequence, indicating thatrecombination had occurred between the pTM(L1) support plasmid and themutant FLC (11). Therefore, subsequent recovery attempts were performedin which the wild type pTM(L1) support plasmid was replaced with onecontaining the appropriate L mutation present in the FLC being rescued.The recovered rHPIV1 viruses were biologically cloned by two successiverounds of terminal dilution using LLC-MK2 monolayers in 96-well plates.(Costar, Corning Inc.) The presence of the introduced mutation in eachbiologically cloned virus was confirmed by sequence analysis of theRT-PCR product.

Replication of rHPIV1 mutants in LLC-MK2 cells at permissive andrestrictive temperatures. The ts phenotype for each of the rHPIV1 mutantviruses was determined by comparing their replication levels to that ofrHPIV1 wild type virus at 32° C., 35° C., 36° C., 37° C., 38° C., and39° C. as described previously (34). Virus titer, which is expressed asa mean log₁₀ 50 percent tissue culture infectious dose per milliliter(log₁₀ TCID₅₀/ml), was determined in one to three separate experiments.The reduction in titer (log) at each restrictive temperature, determinedby comparison to the titer at permissive temperature of 32° C., wasrecorded for each experiment and the mean reduction was calculated. Thets phenotype was defined as a 100-fold or greater reduction in titercompared to the wild type virus.

Replication of rHPIV1 Mutant Viruses in the Respiratory Tract ofHamsters

Four-week-old Golden Syrian hamsters were inoculated intranasally with0.1 ml L-15 (Invitrogen Corp., Grand Island, N.Y.) containing 10⁶⁰TCID₅₀of a wild type or mutant HPIV 1. Four days later, the nasal turbinatesand lungs were collected as previously described (Newman et al., VirusGenes 24:77-92, 2002). Virus present in the samples was quantified bytitration on LLC-MK2 monolayers at 32° C. Infected cells were detectedon day six post-infection by hemadsorption with guinea pig erythrocytes.The mean titer (log₁₀ TCID₅₀/g) was calculated for each group of sixhamsters. The att phenotype was defined as a 100-fold or greaterreduction in virus titer in either or both anatomical locations comparedto wild type.

Determination of the genetic and phenotypic stability of rHPIV1-Y942Hand rHPIV1-Y942A in LLC-MK2 cells by passage at restrictivetemperatures. rHPIV1 mutants with the original Y942H mutation or withthe Y942A mutation were grown on LLC-MK2 monolayers at 32° C. with aninput inoculum of approximately 0.01 TCID50 per cell until cytopathologywas visible (approximately 5-7 days). The virus in the supernatant wasdiluted 1/1000 and was passed again on LLC-MK2 monolayers at 32° C. Thiswas repeated for a total of 10 passages. Alternatively, the two viruseswere also passaged at increasingly restrictive temperatures as follows:two passages (as described above) at 32° C., two at 35° C., two at 36°C., and two at 37° C., after which undiluted supernatant from the second37° C. passage harvest was passed to LLC-MK2 monolayers at 38° C., andthen passed once at 39° C., for a total of 10 passages.

At each passage level aliquots were frozen for phenotypic and genotypicanalysis. The level of replication and temperature sensitivity of bothrHPIV1-Y942H and rHPIV1-Y942A were determined and compared to rHPIV1 asdescribed above. Sequence analysis of each virus was performed asdescribed above.

Results

Recovery of rHPIV1 Bearing Codon Substitution Mutations at Amino AcidPosition 942 in the L Protein

The Y942H mutation in the L protein of the attenuated HPIV3-cp45 viruswas introduced by reverse genetics into the homologous position inrHPIV1, resulting in a viable virus designated rHPIV1-Y942H (Table 5).This mutation in rHPIV1, as in the original HPIV3-cp45 virus, involved asingle nucleotide substitution (TAC to CAC, substitution underlined).Given the sequences of the possible codons for Tyr (TAT and TAC) and His(CAT and CAC), this amino acid substitution could not be designed toinvolve more than a single nucleotide change. To evaluate the full rangeof possible phenotypes involving this amino acid locus, we preparedadditional mutant cDNAs in which position 942 was changed to each of the18 other possible amino acid assignments. Whenever possible, codons werechosen so as to maximize the number of nucleotide differences comparedto the two possible codons for the wild type Tyr assignment (Table 5).

TABLE 5 Recovery of rHPIV1 bearing codon substitution mutations at aminoacid position 942 in the L protein No. of nucleotide Adventitiouschanges needed to rHPIV1 coding restore wt amino mutant mutations VirusCodon^(a) acid recovered? in L ORF rHPIV1 wild type TAT, 0 + nd^(b) TACrHPIV1-Y942H CAC 1 + none rHPIV1-Y942C^(c) TGC 1 + none rHPIV1-Y942F TTT1 + none rHPIV1-Y942N^(c) AAC 1 + none rHPIV1-Y942D^(c) GAC 1 + nonerHPIV1-Y942 S AGC 2 + none rHPIV1-Y942W TGG 2 + none rHPIV I-Y942Q CAG2 + S1302N rHPIV I-Y942K AAA 2 −^(d) nd rHPIV1-Y942I ATC 2 − ndrHPIV1-Y942E GAG 2 − nd rHPIV1-Y942M^(c) ATG 3 + L1367S rHPIV1-Y942A GCG3 + none rHPIV1-Y942T^(c) ACA 3 + none rHPIV1-Y942G GGG 3 + nonerHPIV1-Y942V^(c) GTG 3 + G1755Q rHPIV1-Y942L^(c) CTG 3 + nonerHPIV1-Y942R CGG 3 − nd rHPIV1-Y942P CCG 3 − nd ^(a)In the case of wildtype, the two possible codons yielding the wild type amino acid fortyrosine (Y) are shown; for each mutant the codon chosen forintroduction into rHPIV1 is shown. ^(b)nd, not done: the L gene sequencewas not confirmed either because the virus is the previously-sequencedwild type, or the virus was not recovered. ^(c)Virus was recovered withpTML containing the indicated codon substitution. The other viruses wererecovered using wild type pTML. ^(d)−, not recovered.

Of these 18 additional codon substitution mutants, 13 were recovered ininfectious virus. If the desired rHPIV1 recombinant was not recoveredafter three to five attempts, we considered the mutant to be nonviable.Each of the recovered viruses was biologically cloned and the complete Lgene was sequenced, confirming in each case the presence of theintroduced mutation. Of the 14 recovered rHPIV1 codon substitutionmutants, including the original Y942H mutant, three were found to eachhave one additional adventitious coding mutation in L (Table 5). It isnot unusual to find adventitious mutations, often phenotypically silent,in cloned biologically-derived or recombinant virus when the extraexpedient is taken to perform the extensive sequence analysis involved.The possible contribution of these adventitious mutations to phenotypesexhibited by these three mutants was not further studied because the 11mutants lacking adventitious mutations in the L ORF were more thansufficient for analysis for the purposes of this study. Each of the 14rHPIV1-942 codon substitution mutants replicated efficiently in vitro at32° C. and achieved titers of at least 10⁷ TCID₅₀/ml (Table 6).

TABLE 6 Temperature sensitivity and attenuation phenotype of rHPIV1 Lprotein codon-942 substitution mutants No. of nucleotide Replication ofvirus in hamsters Changes Mean titer of virus needed to (log₁₀ TCID₅₀/restore wild Virus titer at Mean log₁₀ reduction in virus titer at g ±S.E.) type amino 32° C. (log₁₀ indicated temperature (° C.)^(a) No. ofNasal Virus acid TCID₅₀lml) 35 36 37 38 39 animals Turbinates LungsrHPIV1 wild type 0 7.8 0.1 0.4 0.8 1.5 1.8 30   4.2 ± 0.2   3.9 ± 0.4rHPIV1-Y942Hcp45 1 8.0 — 1.4 3.1 5.1 5.8 18   ≦1.5 ± 0.0^(c) ≦1.5 ± 0.0rHPIV1-Y942C 1 8.0 1.8 2.1 2.3 4.8 6.5 6   4.4 ± 0.2   3.4 ± 0.4rHPIV1-Y942F 1 8.1 0.3 1.3 1.9 3.4 5.4 6   3.8 ± 0.1   3.9 ± 0.2rHPIV1-Y942N 1 9.0 1.0 2.1 3.7 4.3 7.0 6   2.0 ± 0.2   1.6 ± 0.1rHPIV1-Y942D 1 7.9 3.3 5.3 >6.6 >6.7 >6.9 6   1.6 ± 0.1   1.8 ± 0.3rHPIV1-Y942S 2 8.3 0.6 1.2 3.6 5.2 >6.4 6   3.2 ± 0.1   1.8 ± 0.2rHPIV1-Y942W 2 8.4 0.8 1.5 3.0 4.2 >6.4 12   1.8 ± 0.2   1.6 ± 0.1rHPIV1-Y942Q 2 7.0 1.4 3.6 >4.7 >5.8 >5.9 12   1.5 ± 0.1   1.7 ± 0.2rHPIV1-Y942M 3 7.3 3.0 5.3 >6.0 >5.8 >6.2 6 ≦1.5 ± 0.0 ≦1.5 ± 0.0rHPIV1-Y942A 3 8.1 3.5 3.5 >5.1 >6.4 >6.8 6 ≦1.5 ± 0.0 ≦1.5 ± 0.0rHPIV1-Y942T 3 8.0 1.8 3.0 >5.0 >6.8 >6.9 6 ≦1.5 ± 0.0 ≦1.5 ± 0.0rHPIV1-Y942G 3 8.1 1.0 3.1 >4.1 >5.8 >7.3 6 ≦1.5 ± 0.0 ≦1.5 ± 0.0rHPIV1-Y942V 3 7.0 2.6 5.0 >5.7 >5.8 >6.1 12 ≦1.5 ± 0.0   1.6 ± 0.1rHPIV1-Y942L 3 7.0 2.8 5.0 5.5 5.5 >5.8 6 ≦1.5 ± 0.0 ≦1.5 ± 0.0^(a)Reduction is compared to titer at 32° C. Values represent average of3 experiments. ^(b)Values in bold are the temperatures at which thevirus titer was reduced 100-fold or more than that of rHPIV1.^(c)Underlined values represent greater than 100 fold reduction comparedto rHPIV1.

Temperature Sensitivity and Attenuation Phenotype of rHPIV1 Codon 942Substitution Mutants

The rHPIV1 virus bearing the Y942H mutation transferred from HPIV3-cp45was strongly ts in vitro, exhibiting a 2.6 log₁₀ reduction in virusyield at 35° C. relative to 32° C. (Table 6). This demonstrates thatthis “imported” mutation functions efficiently as a ts mutation in therHPIV1 backbone. Remarkably, each of the 13 other recovered rHPIV1mutants also was ts, and a spectrum of temperature sensitivity was seenamongst the mutants. Five rHPIV1 mutants, namely those with the Y942D,Y942M, Y942A, Y942V, or Y942L mutation, were found to be as ts asrHPIV1-Y942H, and the latter four involved codons which would eachrequire three nucleotide changes to restore a Tyr at position 942. Thus,a virus with a ts phenotype comparable to that of rHPIV1-Y942H can bereadily generated according to the teachings herein that requires threenucleotide changes at position 942 to restore the wild type Tyr residue.

The ability of the codon substitution viruses to replicate in the upper(nasal turbinates) and lower (lungs) respiratory tract of infectedhamsters was compared to that of wild type rHPIV1 (Table 6). The rHPIV1bearing the Y942H mutation transferred from HPIV3-cp45 was highlyattenuated in both the upper and lower respiratory tract of hamsterscompared to its rHPIV1 parent virus (Table 6). This demonstrates thatthis HPIV3-derived mutation functions efficiently as an att mutation inthe HPIV1 backbone. Eleven of the 13 rHPIV1 mutants were attenuated inhamsters, as defined by exhibiting 100-fold or greater decrease in virustiter in either the upper or lower respiratory tract compared to HPIV1wild type (underlined values in Table 6). Each of the six rHPIV1 mutantsthat would require three nucleotide changes in codon 942 to restore thewild type amino acid (Y942M, Y942A, Y942T, Y942G, Y942V and Y942L) wasas attenuated as rHPIV1-Y942H. Thus, a virus with a comparable attphenotype as the rHPIV1-Y942H can be produced according to the inventionthat requires three nucleotide changes at position 942 to restore thewild type Tyr.

Passage of rHPIV1 Mutants with the Original Y942H Mutation or the“Stabilized” Y942A Codon at Restrictive Temperatures

For ts viruses, passage of the virus at restrictive temperature has beenan effective method to determine the level of stability of the tsphenotype (Belshe et al., J Virol 24:8-12, 1977; Richardson et al., ArchVirol 54:53-60, 1977; Treanor et al, J Virol 68:7684-8, 1994).Therefore, this procedure was employed to compare the stability of thets phenotype of the original mutant (rHPIV1-Y942H), containing a singlenucleotide substitution at codon 942, with that of rHPIV1-Y942A,containing three nucleotide substitutions in this codon. The two viruseswere (i) passaged 10 times at the permissive temperature of 32° C., or(ii) passaged at increasingly restrictive temperatures as follows: twiceat 32° C., twice at 35° C., twice at 36° C., twice at 37° C., once at38° C. and once at 39° C., for a total of 10 passages. Aliquots of virusfrom various passage levels were analyzed for ts phenotype and weresubjected to partial or complete sequence analysis of the L gene (Table7).

TABLE 7 Temperature sensitivity and sequence analysis of rHPIV1 mutantswith the L protein Y942H mutation (single nucleotide substitution) orthe Y942A mutation (three nucleotide substitutions) following passage atrestrictive temperatures Mean titer Mean log₁₀ reduction in Sequence ofL gene and protein^(c) Passage level (log₁₀ virus titer at codon 942Passage analyzed^(b) pfu/ml) indicated temperature (amino acid Extent ofSecond-site Virus series^(a) (° C.) at 32° C. 38° C. 39° C. assignment)sequencing^(d) Mutations rHPIV1 wild 8.0 1.2 3.4 TAT (Tyr) F None typerHPIVI-Y942H unpassaged 6.7 3.7 ^(e) >5.2^(f) CAC (His) C NonerHPIV1-Y942H 1 p10-32 7.7 3.5 5.5 CAC (His) C None rHPIV1-Y942H 2 p4-357.5 3.3 5.3 CAC (His) C None rHPIV1-Y942H 2 p6-36 7.2 2.0 4.0 TAC (Tyr)C None rHPIV1-Y942H 2 p8-37 7.0 0.3 3.5 TAC (Tyr) C None rHPIV1-Y942H 2p9-38 6.5 1.0 3.3 TAC (Tyr) F None rHPIV1-Y942H 2 p10-39 5.2 1.0 3.0 TAC(Tyr) F None rHPIV1-Y942A unpassaged 6.5 4.8 >5.0^(f) GCG (Ala) P NonerHPIV1-Y942A 3 p10-32 7.5 4.0 4.5 GCG (Ala) P None rHPIV1-Y942A 4 p4-357.0 4.3 >5.5 ^(f) GCG (Ala) P None rHPIV1-Y942A 4 p6-36 7.0 4.0 4.8 GCG(Ala) P V1016L rHPIV1-Y942A 4 p8-37 6.7 3.5 >5.2^(f) GCG (Ala) PV1016L/N1125D^(g) rHPIV1-Y942A 4 p9-38 6.2 3.5 >4.7^(f) GCG (Ala) FV1016L/N1125D rHPIV1-Y942A 5 p10-39 5.2 3.0 >3.7^(f) GCG (Ala) F S1328P^(a)Series 1 and 2 involve the Y942H mutant at the permissive andrestrictive temperature regimens, respectively (Materials and Methods).Series 3 involves the Y942A mutant at the permissive regimen, while 4and 5 are independent parallel series at the restrictive regimen.^(b)Samples were analyzed from various passage levels within series 1 to5: each passage level is identified by its number (p1 to p10) and itstemperature (32 to 39, representing 32° C. to 39° C.). ^(c)Consensussequence determined from uncloned RT-PCR products. ^(d)The amount of theL gene sequenced was: F (full), the entire L gene ORF; P, partial,nucleotides10,000-13,300, C, codon-proximal, the region immediatelysurrounding codon 942. ^(e)Value in bold is the temperature at which thevirus titer was reduced 100-fold or more than that of rHPIV1 wild typevirus. ^(f)No detectable virus. ^(g)Electropherogram indicated 50% ofeach nucleotide (A or G) at nucleotide 12143 in codon 1125.

The ts phenotype of each of the two mutant viruses, rHPIV1-Y942H andrHPIV1-Y942A, was unchanged by 10 passages at 32° C. (passage series 1and 3, respectively, in Table 7), and sequence analysis of each L geneindicated that the respective mutant codon was unchanged, and noadditional mutations were detected. In contrast, analysis of aliquotsfrom the passage of the rHPIV1-Y942H mutant (passage series 2 in Table7) at restrictive temperature showed that by passage level p6-36 (36°C.) the virus had lost its ts phenotype and the consensus sequence atcodon 942 had reverted directly back to that of the wild type assignmentof Tyr (CAC to TAC). This single nucleotide change restored the abilityof Y942H-p6 to replicate at restrictive temperatures rendering itindistinguishable from the rHPIV1 wild type virus in this regard. Therewere no other mutations in the L gene of this virus even followingadditional passage at increasingly restrictive temperatures, asconfirmed by sequence analysis of the complete L gene of virus frompassage levels p9-38 and p10-39 (38° C. and 39° C., respectively,passage series 2, Table 3).

In two independent series of passages, the Y942A mutant did not revertat codon 942 even at the highly restrictive temperatures of 38° C. or39° C. (Table 7, passage series 4 and 5), i.e., the sequence at codon942 remained GCG in all passages sequenced. The level of temperaturesensitivity of virus from passage levels p8-37 and p9-38 (37° C. and 38°C., respectively, Table 7, passage series 4) remained highly ts at both38° C. and 39° C. However, there was a partial loss of the ts phenotype,such that the replication of these isolates was increased about 20-foldat 38° C. (Table 3, passage series 4) compared to the original Y942Amutant. This shift in ts phenotype was associated with the acquisitionof two second-site amino acid point mutations in the L protein, V1016Land N1125D. Nonetheless, the p8-37 and p9-38 viruses remained 200-foldmore restricted in their replication at 38° C. than wild type rHPIV1,and both viruses failed to replicate at 39° C. in the ts assay. Thissuggested that the acquisition of the two second-site mutations in Lpartially suppressed the level of temperature sensitivity specified bythe Y942A mutation. Since the complete Y942A p9-38 genome was notsequenced, it also is possible that one or more extragenic suppressormutations also were present. Similarly, the virus from the p10-39 (39°C.) passage level of an independent passage series (Table 7, passageseries 5) manifested an intermediate level of temperature sensitivity at38° C. between that of the wild type rHPIV1 and the starting Y942Avirus, but remained sufficiently ts that it failed to replicate at 39°C. (Table 7). This partial loss of temperature sensitivity wasassociated with the development of a third second-site mutation in L,namely, S1328P. Thus, a ts mutation involving a single nucleotidesubstitution readily reverted to the wild type assignment and rapidlybecame the predominant viral species during passage at restrictivetemperatures. In contrast, reversion was not observed involving a codonthat differed from wild type by three nucleotides, but a partial loss ofthe ts phenotype was observed after 8 passages at increasinglyrestrictive temperatures, and putative second-site intragenic suppressormutations were detected.

Recovery of rHPIV1 Bearing Codon Substitution Mutations at Amino AcidPosition 992 in the L Protein

A second L protein mutation from HPIV3-cp45 L, L992F, was introducedinto the homologous position in rHPIV1 by reverse genetics, resulting ina viable virus designated rHPIV1-L992F (Table 8). Additional mutantswere constructed in which position 992 was changed into 17 of the 18other possible amino acid assignments (lacking only Ser, Table 8).Codons were chosen to maximize the number of nucleotide differencescompared to the possible codons for the wild type assignment of Leu, butthis was made difficult by the existence of six different codons forLeu. Nonetheless, nine of the substitution mutations could be designedto involve two nucleotide differences compared to any Leu codon (Table8). Of these 17 additional mutations, 10 recombinant viruses bearing theappropriate mutation were recovered and were readily propagated in vitroand were biologically cloned. Of the seven remaining rHPIV1 mutants,three (L992R, L992P, and L992Q) were recovered in the transfectionharvest but reverted to wild type during biological cloning, two others(L992E and L992D) were recovered in the transfection harvest butreplicated very inefficiently and could not be propagated. Two others(L992G and L992T) recombined with the wt pTM(L1) and were not furtherstudied. The L gene was sequenced around the site of the mutation ineach of the recovered, stable mutant viruses, and the presence of eachintroduced mutation was confirmed. Each of the recovered codon 992substitution mutants replicated efficiently in vitro at 32° C. andachieved titers of ≧1≧10⁷ TCID₅₀/ml (Table 9).

TABLE 8 Recovery of rHPIV1 bearing codon substitution mutations at aminoacid position 992 in the L protein No. of nucleotide changes needed torHPIV1 restore wild type mutant Virus Codon^(a) amino acid recovered?rHPIV1 wild type TTA, TTG, CTT, 0 + CTC, CTA, CTG rHPIV1 L992Fcp45 TTT1 + rHPIV1 L992M ATG 1 + rHPIVI L992H CAC 1 + rHPIV1 L992I ATC 1 +rHPIV1 L992W TGG 1 + rHPIV1 L992R CGG 1 −^(b) rHPIV1 L992V GTC 1 +rHPIV1 L992Q CAG 1 −^(b,c) rHPIV1 L992E GAG 1 −^(d) rHPIV1 L992P CCG 1−^(b) rHPIV1 L992A GCG 2 + rHPIV1 L992Y TAC 2 + rHPIV1 L992C TGC 2 +rHPIV1 L992N AAC 2 + rHPIV1 L992D GAC 2 −^(d) rHPIV1 L992T ACC 2 −^(c)rHPIV1 L992G GGG 2 −^(c) rHPIV1 L992K AAG 2 + ^(a)In the case of wildtype, the six possible codons yielding the wild type amino acid forleucine (L) are shown; for each mutant the codon chosen for introductioninto rHPIV1 is shown. A cDNA bearing an L992S mutation was notconstructed. ^(b)Mutant was recovered but reverted to wild type duringpassage at 32° C. and was not further studied. ^(c)Mutant recombinedwith wild type pTML₁ and was not further studied. ^(d)Virus wasrecovered from transfection, but titer was low and virus was lost onsubsequent passage.

TABLE 9 Temperatire sensitivity and attenuateion phenotype of rHPIV1 Lprotein codon-992 substition mutants Replication of virus in hamstersNo. of nucleotide Mean log₁₀ reduction^(a) Mean titer of virus (log₁₀changes needed Virus titer at in virus titer at TCID₅₀/g ± S.E.) torestore wild 32 C. (log₁₀ indicated temp (° C.) No. of Nasal Virus typeamino acid TCID₅₀/ml)b 35 36 37 38 39 animals Turbinates LungsrHPIV1-wild type 0 7.5 0.1 0.1 0.5 0.6 1.2 30 4.2 ± 0.2 3.9 ± 0.4 rHPIV1L992Fcp45 1 8.1 0.2 0.3 1.3 0.9 2.4 12 4.4 ± 0.3 3.0 ± 0.2 rHPIV1 L992M1 8.4 0.2 0.2 0.4 0.7 2.7 6 4.7 ± 0.3 3.2 ± 0.4 rHPIV1 L992I 1 8.2 0.30.5 0.5 2.7 4.3 ^(b) 6 4.1 ± 0.2 2.6 ± 0.3 rHPIV1 L992W 1 8.5 0.0 0.50.8 3.7 3.8 6 4.0 ± 0.2 3.3 ± 0.1 rHPIV1 L992H 1 8.1 0.3 1.3 1.3 1.8 4.96 3.3 ± 0.2 3.4 ± 0.1 rHPIV1 L992V 1 6.2^(d) 0.7 0.5 1.5 2.5 4.0 6≦1.5 ± 0.0^(c)   1.7 ± 0.2 rHPIV1 L992N 2 7.8 0.6 0.3 0.6 2.0 3.3 6 2.7± 0.2 ≦1.5 ± 0.0  rHPIV1 L992Y 2 8.5 0.0 0.2 0.5 1.4 2.9 6 4.5 ± 0.2 3.3± 0.4 rHPIV1 L992K 2 8.6 0.0 1.2 0.2 1.8 3.0 6 4.1 ± 0.3 3.1 ± 0.4rHPIV1 L992A 2 7.9 0.5 0.3 1.2 3.0 4.5 6 3.6 ± 0.2 3.2 ± 0.3 rHPIV1L992C 2 7.3 0.7 2.2 2.9 4.0 >4.9 6 3.2 ± 0.2 1.7 ± 0.1 ^(a)Reduction iscompared to titer at 32° C. Values represent average of 2-3 experiments.^(b)Values in bold are the temperatures at which the virus titer wasreduced 100-fold or more than that of rHPIV1. ^(c)Underlined valuesrepresent greater than 100-fold reduction compared to rHPIV1 in the samepulmonary compartment. ^(d)Other preparations of this virus had titersof ≧10⁷, indicating efficient growth at permissive temperature.

Temperature Sensitivity and Attenuation Phenotype of rHPIV1 codon 992Substitution Mutants

The rHPIV1 virus bearing the L992F mutation transferred from HPIV3-cp45was not ts in vitro (Table 9). Six of the 10 additional rHPIV1 mutantsthat were recovered were ts, and a spectrum of temperature sensitivitywas seen amongst the mutants. One virus, rHPIV1-L992C, exhibited thegreatest level of temperature sensitivity, with a 100-fold reduction ofreplication at 36° C. Thus, a rHPIV1 codon 992 substitution mutant wasgenerated according to the teachings herein that, in contrast to theoriginal non-ts rHPIV1-L992F mutant, was substantially ts.

The level of replication of the codon 992 substitution mutants in theupper and lower respiratory tract of hamsters was compared with that ofrHPIV1 (Table 9). The rHPIV1-L992F virus was not attenuated in hamsters:thus, the L992F mutation is attenuating in HPIV3 (Skiadopoulos et al., JVirol 72:1762-8. 1998) but not HPIV1. However, three of the 10 otherrecovered codon 992 substitution mutants (L992V, L992N, and L992C) werereduced in replication 100-fold or more compared to wild type virus.Thus, several rHPIV1 codon 992 substitution mutants were providedaccording to the teachings herein with an att phenotype. Interestingly,the rHPIV1-L992V mutant was only slightly ts, suggesting that the L992Vmutation confers a predominantly non-ts attenuation phenotype.

The ability to recover recombinant negative strand RNA viruses from cDNAmakes it possible to develop live attenuated virus candidates for use inimmunogenic compositions by the planned introduction of attenuatingmutations to a wild type virus. This can be done in a sequential mannerin response to ongoing pre-clinical and clinical evaluation until adesired balance between attenuation and immunogenicity is achieved(Collins et al., Virology 296:204-11, 2002; Murphy et al., J Clin Invest110:21-7, 2002). Within the present invention, genetic stability ofrHPIV1 and rHPIV2 can be achieved by the accumulation of a sufficientnumber of attenuating mutations to make loss of the attenuationphenotype unlikely during manufacture and during the brief period ofreplication of these respiratory viruses in humans (Collins et al.,Virology 296:204-11, 2002; Murphy et al., J Clin Invest 110:21-7, 2002).In addition, attenuating mutations can be designed that have improvedstability, such as the deletion of entire genes or, as in the presentstudy, the “stabilization” of point mutations. The addition of pointmutations that specify the ts and att phenotype to partially attenuatedviruses is useful in the incremental attenuation of viruses such asrespiratory syncytial virus and HPIV3 (Collins et al., Virology296:204-11, 2002; Skiadopoulos et al., Virology 260:125-35, 1999).However, as indicated above, the att phenotype specified by thesemutations can be subject to modification by direct reversion or othermutation during replication in vivo (Tolpin et al., Virology112:505-517, 1981; Wright et al., J Infect Dis 182:1331-1342, 2000).Since molecular engineering makes it possible to alter codons thatspecify the ts and att phenotypes, the present study was useful toelucidate whether the choice of alternative codons at a given locus canbe used to enhance the genetic stability of a ts-att mutation and toaugment the level of attenuation.

The introduction of the Y942H mutation of the L gene of HPIV3-cp45 intothe corresponding position of rHPIV1 yielded a virus that possessed thets and att phenotypes. However, these phenotypes were the result of asingle nucleotide substitution in codon 942 and thus might be subject toinstability. Thirteen additional viable rHPIV1 mutants were generatedthat contained various alternative amino acid assignments at codon 942,and all were ts. The noted failure to recover 5 amino acid substitutionmutants indicates that mutations that specify these amino acids in codon942 are dead-end mutations, i.e., they would be non-viable. Nonetheless,eleven of the 13 recovered, viable mutants were successfullydemonstrated to be attenuated for replication in the respiratory tractof hamsters. Six highly attenuated mutants (those with a Met, Ala, Thr,Gly, Val, or Leu substitution at position 942) were identified thatwould require three nucleotide changes to occur to generate a codon thatspecified the wild type Tyr residue at codon 942. However, two othermutants (those with a Cys or Phe at position 942) out of the 13recovered rHPIV1 mutants replicated in hamsters as efficiently as wildtype HPIV1. Thus, the naturally-occurring Tyr-942 assignment was not theonly one that conferred a wild type-like phenotype: Cys-942 and Phe-942did as well.

Therefore, the present invention provides additional direction andguidance to choose a 942 assignment that (i) specifies an appropriatelevel of attenuation, and (ii) differs by two, or preferably three,nucleotides from all possible codons for Tyr, Cys and Phe. For four ofthe six highly attenuated viruses (those with a Met, Gly, Val, or Leusubstitution), it would take only two nucleotide substitutions in theircodons to give rise to a virus with a Tyr, Cys, or Phe at position 942.The remaining two highly attenuated viruses (with Ala or Thrassignments) would take three nucleotide substitutions to generate acodon for Tyr, Cys, or Phe. Thus, in this systematic examination of thets and att phenotypes of 13 viable codon 942 substitution mutations, theY942A and the Y942T mutations were identified as codon 942 substitutionsthat were highly attenuated and would require three nucleotide changesto occur to yield a virus with a wild type-like phenotype. Fortunately,both of these viruses were among the 11 mutants that were recoveredwithout adventitious mutations in the L gene, and thus these findingsare unambiguous.

The genetic and phenotypic stability of one of these two rHPIV1 viruses,the Y942A virus, was examined following replication at restrictivetemperatures. This was done in parallel with the original Y942H virus,which differed from wild type by only a single nucleotide. The Y942Hvirus readily reverted to wild type phenotype after only four passagesat 35-36° C., whereas the “codon stabilized” Y942A virus exhibited onlya partial loss of the ts phenotype even after eight passages at elevatedtemperatures. The phenotypic instability of the Y942H virus wasaccompanied by a direct reversion to the wild type sequence at codon942. This change occurred only following replication at elevatedtemperature, demonstrating the important role that elevated temperatureplayed in selecting for the ts rHPIV1-Y942H revertant.

Sequence analysis confirmed that the “stabilized” Y942A virus retainedthe GCG Ala codon throughout its passages at elevated temperatures.However, the partial loss of the ts phenotype noted above was associatedwith the acquisition of one or two second-site amino acid substitutionsinvolving three different positions in L. This suggests that these areintracistronic suppressor mutations that partially restored the abilityof rHPIV1-Y942A to replicate at 38° C. ft also is possible that anextracistronic suppressor mutation, e.g., in P or N, could havedeveloped that contributed to the partial restoration of replication ofthe mutant at elevated temperature (Mucke et al., Virology 158:112-7,1987; Treanor et al, J Virol 68:7684-8, 1994). The finding that the“stabilized” Y942A virus retained a high degree of temperaturesensitivity even following passage at highly restrictive temperaturessuggests that it also would be more phenotypically stable in vitro andin vivo than the “non-stabilized” Y942H virus. This analysis of thecodon substitutions at residue 942 of the HPIV1 L protein provides anexample of how to identify mutations that exhibit a set of propertiesincluding both attenuation and phenotypic/genetic stability desirablefor inclusion in a live attenuated virus. This particular example turnedout to have several advantages that facilitated the selection of a“stabilized” mutant assignment: (i) the wild type assignment of Tyr hasonly two possible codons, and (ii) among the mutants with alternativeamino acid assignments, only two were not highly attenuated, and (iii)these two assignments each has only two possible codons.

It was previously reported that the introduction of the HPIV3-cp45 L992FL protein mutation into a wild type HPIV3 backbone conferred the tsphenotype in vitro and the att phenotype in hamsters (30), identifyingL992F as an independent ts att mutation. However, the introduction ofthis mutation into the homologous position in the HPIV1 background inthe present study did not confer either phenotype. When alternativeamino acid assignments were introduced at codon 992, only 10 of 17mutants were recovered, indicating that this site is indeed sensitive tomutation. Three codon substitution mutants were identified,rHPIV1-L992V, rHPIV1-L992N, and rHPIV1-L992C, that were 10- to 100-foldrestricted in replication in the upper or lower respiratory tract ofhamsters compared to rHPIV1 wild type virus. Thus, these findingsexemplify a second use of codon substitution mutations, i.e., togenerate mutants with an enhanced level of attenuation in vivo. It wasof interest to find that each of the three attenuated recombinants(rHPIV1-L992C, rHPIV1-L992V and rHPIV1-L992N) also was temperaturesensitive. However, there was a dissociation between the level oftemperature sensitivity and the level of attenuation. For example theL992C mutation restricted replication in vitro at 36-37° C. and wasattenuating primarily in the lower respiratory tract, whereas the L992Vmutation restricted replication in vitro only at 39° C. but was highlyattenuating in both the upper and lower respiratory tract. Thus, theL992V mutation is predominantly of the non-ts type. This is in contrastto the situation with the codon 942 substitution mutations examined inthe present study, where there generally was a strong associationbetween the level of temperature sensitivity in vitro and the level ofattenuation in vivo.

Live attenuated viruses that contain ts attenuating mutations can bestabilized by the addition of non-ts attenuating mutations (Murphy etal., Vaccine 15:1372-1378 1997). Also, ts mutations exert theirattenuating effect more prominently in the lower, warmer region of therespiratory tract, whereas non-ts mutations would attenuate irrespectiveof this temperature gradient. Hence, a mixture of both types ofmutations would be optimal to achieve the dual goals of preventingserious lower respiratory tract disease and reducing upper respiratorytract congestion. The ts and non-ts codon substitution mutationsidentified in this report are now being combined with additional non-tsattenuating mutations to generate rHPIV1 virus that are satisfactorilyattenuated, efficacious in immunogenic compositions, and geneticallystable.

EXAMPLE IX Production and Characterization of Recombinant HPIV1 P/C GeneDeletion Mutants

In additional aspects of the invention, the recombinant HPIV1 or HPIV2genome or antigenome comprises an additional nucleotide modificationspecifying a phenotypic change selected from a change in growthcharacteristics, attenuation, temperature-sensitivity, cold-adaptation,plaque size, host-range restriction, or a change in immunogenicity. Forexample, in HPIV1 these additional modifications can alter one or moreof the N, P, C, C′, Y1, Y2, M, F, HN and/or L genes and/or a 3′ leader,5′ trailer, a cis-acting sequence such as a gene start (GS) or gene end(GE) sequence, and/or intergenic region within the HPIV1 genome orantigenome. For example, one or more HPIV1 gene(s) can be deleted inwhole or in part, or expression of the gene(s) can be reduced or ablatedby a mutation in an RNA editing site, by a frameshift mutation, by amutation that alters a translation start site, by introduction of one ormore stop codons in an open reading frame (ORF) of the gene, or by amutation in a transcription signal. In specific embodiments, therecombinant HPIV1 genome or antigenome is modified by a partial orcomplete deletion of one or more C, C′, Y1, and/or Y2 ORF(s) or otherauxiliary gene, or one or more nucleotide change(s) that reduces orablates expression of one or more of the C, C′, Y1, and/or Y2 ORF(s) orother auxiliary gene. In other embodiments, the recombinant HPIV1 genomeor antigenome is modified to encode a non-PIV molecule selected from acytokine, a T-helper epitope, a restriction site marker, or a protein ofa microbial pathogen capable of eliciting an immune response in amammalian host.

Interferons, which are host cell proteins elaborated in response toinfection with viruses, induce an antiviral state in cells thatrestricts replication of virus in the interferon treated cells. Sincethis is a powerful component of the host's innate immunity, it is notsurprising that many viruses have developed elaborate strategies tocounteract the antiviral activity of the interferons (Garcia-Sastre,Virology 279:375-384, 2001; Goodbourn et al., J. Gen. Virol.81:2341-2364, 2000; Samuel, Clin. Microbiol. Rev. 14:778-809, 2001). TheC and V proteins of many paramyxoviruses, which are encoded byalternative translational open reading frames (ORFs) in the P gene ofthe paramyxoviruses (Chanock et al., In Fields Virology 1:1341-1379,2001), are involved in inhibition of the host-cell response to both Type1 and Type 2 interferons. Mutations that affect the C or V ORFs of PIV1or PIV2 viruses often result in ablation of this anti-interferonactivity (Didcock et al., J. Virol. 1999; Garcin et al., J. Virol.75:6800-6807, 2001; Garcin et al., Virology 295:256-265, 2002; Parisienet al., Virology 283:230-239, 2001), and viruses with such mutationsbecome sensitive to antiviral actions of interferon and exhibit reducedreplication in vitro in interferon competent cells and in vivo ininterferon competent animals (Garcia-Sastre, Virology 279:375-384,2001). Viruses with such mutations have been considered for use as liveattenuated virus vaccines (Garcia-Sastre, Virology 279:375-384, 2001),since they can readily be prepared in vitro in known interferon-negativecells. The V and C proteins have functions other than just putativeinterferon function (Chanock et al., In Fields Virology 1:1341-1379,2001); Lamb et al., In Fields Virology 1:1305-1340, 2001), therefore,introduced mutations could affect one or more of the functions of theaccessory proteins. Since the complete set of the functions of theaccessory proteins have not been defined, mutations in the accessoryproteins that attenuate the virus might do so by a mechanism that is notrelated to its anti-interferon properties. Thus, a goal in developingimmunogenic compositions of the invention includes production of liveattenuated HPIV1 whose attenuation is based solely, or in part, on thepresence of mutations that render the virus fully susceptible to thehost's interferon response.

Since HPIV1 lacks a V ORF (Newman et al., Virus Genes 24:1, 77-92,2002), the anti-interferon protein of this virus may be one or more ofthe C proteins (including the set of C, C′, Y1, and Y2 proteins).Mutations in the C protein of Sendai virus, a murine PIV1 highly relatedto HPIV1, that interfere with the antiviral activity of interferon andthat attenuate the replication of this virus for mice have beendescribed (Garcin et al., J. Virol. 75:6800-6807, 2001; Garcin et al.,Virology 295:256-265, 2002). Single-nucleotide substitution mutationsthat affect the C protein, but not the P protein, in recombinant HPIV3have been reported (Durbin et al., Virology 261:319-330, 1999;Skiadopoulos et al., J. Virol. 73:1374-1381, 1999a). HPIV3 recombinantsbearing the HPIV3 cp45 C mutation (196T) or the F170S mutation werereportedly restricted for replication in vivo but not in vitro and,similarly, rHPIV1 bearing the F170S_(MPIV1) mutation in C was reportedlyattenuated in hamsters. These mutants were not is and replicatedefficiently in vitro. These types of non-ts attenuating mutations are animportant element in the production of phenotypically stablelive-attenuated viruses of the invention, as outlined herein. However,only a single-nucleotide substitution specifies the F170S mutation, andsuch mutations would therefore require only a single nucleotidesubstitution to revert to a wild type virulence phenotype. The findingssummarized in the present Example present a method to produce liveattenuated rHPIV1 subviral particles that contain functional deletionsin the C protein, which will exhibit greater stability of theattenuation phenotype in vivo. Also described is the recovery of rHPIV1viruses bearing these deletion mutations.

To generate live-attenuated HPIV1 recombinants whose likelihood torevert to wt is highly diminished, deletion mutations were introducedwithin the P/C gene of HPIV1 in the region of the overlap of the P and CORFs. A region located in the N-terminal end of the HPIV1 C protein thatmay interact with and abrogate the cell's interferon response (Garcin etal., J. Virol. 75:6800-6807, 2001) pathway was mutagenized. Mutationswere introduced in this area by PCR mutagenesis that deleted codons10-15 of the C ORF. This mutation also deleted codons 13-19 of the PORF. A subset of mutations deleting C ORF codons 10-11, 12-13, and 14-15were also generated by PCR mutagenesis (see FIG. 13 of U.S. patentapplication Ser. No. 10/302,547, filed by Murphy et al. on Nov. 21,2002; and corresponding PCT Publication Number WO 03/043587 A2,published on May 30, 2003, each incorporated herein by reference).Preferable mutants are ones in which C function is altered withoutaffecting P function, since the latter is an essential protein requiredfor viable HPIV1. Therefore, we first evaluated the ability of a P genecontaining these mutations to support the recovery of rHPIV1 from afull-length rHPIV1 antigenomic cDNA in transfected cells. This is anappropriate assay for P function, since a functional P support plasmidis an essential component of the set of three support plasmids used inthe recovery of infectious viruses from transfected infectiousparainfluenza virus cDNAs. Each of the five deletion mutations indicatedwere introduced into the pTM-(P₁) support plasmid. HEp-2 cells weretransfected with pTM (N₁), pTM (L₁), full-length wild type HPIV1antigenomic cDNA, and each of the pTM (P₁) containing the deletionsindicated and were coinfected with MVA-T7, as described above.Surprisingly, each of the P deletion mutants supported the recovery ofrHPIV1 from cDNA. Importantly, infectious rHPIV1 was not recovered fromcontrol transfection reactions lacking a P support plasmid, indicatingthat the pTM (P₁) containing the deletions were functional.

A P/C gene deletion mutation specifying deletion of amino acids 10-15 inthe N-terminal end, or at amino acids 168-170 of the C proteins wereintroduced into the full-length antigenomic HPIV1 cDNA, and these cDNAswere used to recover mutant recombinant HPIV1 containing P/C genedeletions. Two viruses have been recovered to date, and they grew tohigh titer in cell culture indicating that the introduced mutations werenot attenuating in vitro. rHPIV1 C: Δ10-15(F_(RSV)), a recombinant HPIV1expressing the RSV F protein from a supernumerary gene inserted upstreamof the HPIV1 N ORF and encoding a deletion of C protein amino acids10-15 grew to 7.0 log₁₀ TCID₅₀/ml in LLC-MK2 cells. Thus, this rHPIV1replicates efficiently in tissue culture despite a six amino aciddeletion in P, a six amino acid deletion in C, and a six amino aciddeletion in C′. The attenuation of rHPIV1 viruses bearing this mutation,which we have shown to be viable and capable of efficient replication intissue culture cells, can now be readily determined in hamsters andAfrican Green monkeys. If rHPIV1 viruses bearing this mutation areappropriately attenuated and immunogenic, this mutation can beintroduced into any rHPIV1 alone or along with other ts and non-tsattenuating mutations to generate phenotypically stable live-attenuatedrHPIVs.

An additional 2-codon deletion mutation was introduced in the middle ofthe P/C gene. This mutation spans amino acid F170, whose substitution atamino acid residues 168-170 of the rHPIV1 C protein has been shown toconfer a non-ts attenuation phenotype. The mutation was also introducedinto pTM (P₁) and this support plasmid was functional in the rescueassay described above (FIG. 15, Panel B), indicating that the functionof the P protein is not adversely affected. A rHPIV1 virus bearing thedeletion in the P/C gene, designated rHPIV1 C:ΔF170, was recovered fromtransfected cells as described previously. This deletion mutationmodifies each of the 5 known P/C gene proteins (P, C, C′, Y1, and Y2)including deletion of amino acid 172 and 173 in the P protein, and168-170 in the C protein. Although this mutant encoded 5 protein withdeletion, it replicated well in cell culture. Satisfactorily attenuatedand phenotypically stable rHPIV viruses can be generated bearing thisdeletion mutation alone or in combination with other attenuatingmutations for use in immunogenic compositions and methods of theinvention.

Characterization of the Level of Replication and Efficacy Against WildType HPIV1 Challenge of a Recombinant HPIV1 C Protein Deletion Mutant inHamsters and Non-Human Primates

The in vivo growth characteristics of rHPIV1 C:F170S bearing the singleC protein amino acid substitution at F170 and rHPIV1 C:ΔF170 containingthe P/C gene deletion mutation described above was examined in animalmodels generally accepted as predictive of HPIV replicative potentialand immunogenic activity in humans, namely Golden syrian hamsters(Mesocritus Auratus) and African green monkeys (Cercopithecus aethiops).The level of replication of rHPIV1 C:F170S and rHPIV1 C:ΔF170 in theupper and lower respiratory tract of infected hamsters was compared tothat of unmodified biologically derived (HPIV1_(LLC1)) and recombinantHPIV1 (rHPIV1_(LLC4)) control viruses. As shown in Table 10, the levelof replication of rHPIV1-ΔF170 was highly restricted in both the upperand lower respiratory tract of hamsters and was nearly identical to thatof the rHPIV1 C: F170S bearing the F170S substitution mutation,indicating that deletion of the F170 amino acid results in a similarattenuation phenotype as the F170 to serine substitution mutation. Thisattenuation phenotype is characterized by a similar level of restrictionof replication in both the upper and lower respiratory tracts, a highlydesirable phenotype that should result in abrogation of the upper(rhinitis and otitis media) and lower respiratory tract disease (croup)caused by this virus in humans. Since rHPIV1 C:F170S, rHPIV1 C:ΔF170,and rHPIV1 C: Δ10-15(F_(RSV)) each contain the R84G amino acidsubstitution in the C protein and the T553A substitution in the HNprotein that confer a host range attenuation phenotype on rHPIV1_(LLC4),the present observations indicate that the substitution and deletionmutations at C protein residue 170 and at residues 10-15 of C arecompatible for viability with the host range attenuating mutations,namely, the R84G amino acid substitution in the C protein and the T553Asubstitution in the HN protein. Thus the attenuation phenotype of therHPIV1 mutants containing these sets of attenuating mutations should behighly phenotypically stable in vitro.

TABLE 10 Replication of rHPIV1 wt and mutant viruses in hamstersReplication of immunizing virus in hamsters: No. of Mean virus titerNucleotides (log₁₀ TCID₅₀/ Required for g ± S.E.^(a)) in: ImmunizingReversion to Nasal Virus^(b) WT No. animals Turbinates LungsHPIV1_(LLC1) 0 6 5.1 ± 0.1 4.5 ± 0.5 rHPIV1_(LLC4) 0 6 4.0 ± 0.1 3.6 ±0.3 rHPIV1 C:ΔF170   3+ 6 2.5 ± 0.1^(c) 2.6 ± 0.2^(c) rHPIV1 C:F170S 112 2.5 ± 0.1^(c) 2.5 ± 0.1^(e) ^(a)S.E. Standard error ^(b)Hamsters wereinoculated IN with 10⁶ TCID₅₀ of the indicated virus. Nasal turbinatesand lung tissues from six animals for each group were harvested on day4. Virus present in the tissues was quantified by serial dilution onLLC-MK2 monolayers at 32° C. and infected cultures were detected byhemadsorption with guinea pig erythrocytes after 6 days. ^(c)Values inbold show an approximately 100-fold or more reduction in titer comparedto the titer of wild type HPIV1_(LLC1).

The level of replication of the rHPIV1 C:ΔF170 recombinant was alsocompared to the biologically-derived (rHPIV1_(LLC1)) and recombinantHPIV1 (rHPIV1_(LLC4)) in HPIV1 seronegative African green monkeys by theintranasal (IN) and intratracheal (IT) administration of 106 TCID₅₀ ofvirus at each site, as described previously. As shown in Table 11, themean peak virus titer of the mutant rHPIV1 with the ΔF170 or the F170S Cprotein mutation was approximately 100-fold and 40-fold attenuated forreplication in the upper and lower respiratory tract of infectedmonkeys, respectively, compared to the replication of wild typeHPIV1_(LLC1) virus. The daily mean virus titer for the C protein mutantswas also reduced compared to that of the wild type HPIV1_(LLC1) virusand to rHPIV1_(LLC4) (FIG. 16). The recombinants containing a ΔF170 or aF170S mutation in the C protein were also more attenuated forreplication in the lower respiratory tract compared to the parentrHPIV1_(LLC4) virus, indicating that the attenuation specified by themutations at residue 170 in the C protein are additive to theattenuation specified by the mutations found in the C or HN protein ofrHPIV1_(LLC4) for the lower respiratory tract of African Green monkeys.Thus, both the deletion and substitution mutation in the HPIV1anti-interferon C protein confer an attenuation of replication phenotypein the respiratory tract of African green monkeys. Despite thisattenuation, both viruses induced a high level of protection againstwild type virus (Table 11). Importantly, the ΔF170 mutation wouldrequire an insertion of 6 nucleotides to revert to a wild type HPIV1sequence. Because this recombinant mutant virus was designed to conformto the rule of six, correcting insertions into mutant PIVs such as theseP gene deletion mutants are predicted to be extremely unlikely to occur.Furthermore, C protein deletion mutations such as the one described herewill very likely impart an exceptionally stable attenuation phenotypefollowing replication in vitro and in vivo, an important considerationfor the safety of the immunizing virus in the target infant population,and for the large scale manufacture of immunogenic compositions. Ifnecessary, the various attenuating mutations identified in the C proteincan be combined with each other or with attenuating mutations identifiedin other parts of the genome to modify the level of attenuation ofrecombinant HPIVs of the invention.

TABLE 11 Level of virus replication in seronegative African greenmonkeys infected with biologically derived or recombinant HPIV1 Meanpeak virus Mean peak titer ± S.E.^(b) challenge virus Number (log₁₀titer^(e) (log₁₀ Virus of TCID₅₀/ml) in. TCID₅₀/ml) in: administered^(a)animals NP swab^(c) TL^(d) NP swab TL HPIV1_(LLC1) 4 4.2 ± 0.3 5.1 ± 0.70.8 ± 0.3 0.5 ± 0.0 rHPIV1_(LLC4) 10 2.4 ± 0.3 5.0 ± 0.3 0.6 ± 0.1 0.6 ±0.1 rHPIV1 C: 4 2.0 ± 0.1 3.5 ± 0.6 1.1 ± 0.4 0.5 ± 0.0 F170S rHPIV1 C:4 2.1 ± 1.0 3.6 ± 0.6 0.8 ± 0.1 1.2 ± 0.4 ΔF170 None 4 nd nd 4.7 ± 0.56.2 ± 0.7 ^(a)Monkeys were inoculated intranasally and intratracheallywith 10⁶ TCID₅₀ of the indicated virus. Data was compiled from threestudies. ^(b)Mean of the peak virus titers for the animals in each groupirrespective of sampling day. SE, standard error. Virus titrations wereperformed on LLC-MK2 cells at 32° C., and infected cultures weredetected after 7 days by hemadsorption with guinea pig erythrocytes. Thelimit of detection was 1.0 log₁₀ TCID₅₀/ml. ^(c)Nasopharyngeal sampleswere collected on days 0 to 10 postinfection. The titers on day 0 were≦0.5 log₁₀ TCID₅₀/ml. ^(d)Tracheal lavage samples were collected on days2, 4, 6, 8, and 10 postinfection. The titers on day 0 were ≦0.5 log₁₀TCID₅₀/ml. ^(e)Each animal was challenged with the wild type Wash/64strain, HPIV1_(LLC1). NP and TL samples were collected on days 2, 4, 6,and 8. The titer of virus present in the samples was determined asdescribed above.

EXAMPLE X Use of Recombinant HPIV1 as an Expression Vector forSupernumerary Foreign Genes

In yet additional aspects of the invention, the recombinant HPIV1 genomeor antigenome comprises a partial or complete HPIV1 “vector” genome orantigenome that is combined with one or more heterologous gene(s) orgenome segment(s) encoding one or more antigenic determinant(s) of oneor more heterologous pathogen(s) to form a chimeric HPIV1 genome orantigenome. The heterologous gene(s) or genome segment(s) encoding theantigenic determinant(s) can be added as supernumerary gene(s) or genomesegment(s) adjacent to or within a noncoding region of the partial orcomplete HPIV1 vector genome or antigenome, or can be substituted forone or more counterpart gene(s) or genome segment(s) in a partial HPIV1vector genome or antigenome. The heterologous gene(s) or genomesegment(s) can include one or more heterologous coding sequences and/orone or more heterologous regulatory element(s) comprising an extragenic3′ leader or 5′ trailer region, a gene-start signal, gene-end signal,editing region, intergenic region, or a 3′ or 5′ non-coding region.

In related embodiments of the invention, chimeric HPIV1 viruses areprovided wherein the vector genome is combined with one or moreheterologous antigenic determinant(s) of a heterologous pathogenselected from measles virus, subgroup A and subgroup B respiratorysyncytial viruses, mumps virus, human papilloma viruses, type 1 and type2 human immunodeficiency viruses, herpes simplex viruses,cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses,bunyaviruses, flaviviruses, alphaviruses, human metapneumoviruses, andinfluenza viruses. In exemplary aspects, the heterologous antigenicdeterminant(s) is/are selected from measles virus HA and F proteins,subgroup A or subgroup B respiratory syncytial virus F, G, SH and M2proteins, mumps virus HN and F proteins, human papilloma virus L1protein, type 1 or type 2 human immunodeficiency virus gp160 protein,herpes simplex virus and cytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ,gK, gL, and gM proteins, rabies virus G protein, Epstein Barr Virusgp350 protein, filovirus G protein, bunyavirus G protein, flavivirus preM, E, and NS1 proteins, human metapneuomovirus G and F protein, andalphavirus E protein, and antigenic domains, fragments and epitopesthereof.

The complete genomic sequence of a biologically derived HPIV1/Wash/64strain that had been passaged four times in LLC-MK2 cells and hadacquired two putative host-range amino acid substitutions in the C andHN proteins was determined (GenBank Accession No. AF457102), and afull-length antigenomic HPIV1 cDNA was generated. A recombinant virus(rHPIV1_(LLC4)) that was derived from this cDNA was attenuated forreplication compared to a biologically derived HPIV1 that had beenpassaged once in LLC-MK2 cells (HPIV1_(LLC1)). This antigenomic cDNAencoding a C protein R84G substitution and an HN protein T553Asubstitution was used to derive the recombinant viruses described below.

To generate an antigenomic HPIV1 cDNA that could be used as a vector, aunique Mlu I restriction site was introduced immediately upstream of theHPIV1 N gene translation initiation codon in the full-length antigenomicHPIV1 cDNA (nts 113-118) by PCR mutagenesis, as described previously(U.S. patent application Ser. No. 10/302,547, filed by Murphy et al. onNov. 21, 2002; and corresponding PCT Publication Number WO 03/043587 A2,published on May 30, 2003, each incorporated herein by reference). Thesupernumerary gene insertion site in the vector was designed so that itdid not disrupt any of the postulated HPIV1 replication andtranscription cis-acting elements predicted by analogy to heterologousparamyxoviruses. The present example describes insertion of anadditional transcriptional unit into the MluI site (nts 113-118)upstream of the N protein ORF (FIG. 14). However, based on thesuccessful results described herein, alternative unique restrictionsites can also be used, and these can also be introduced at other genejunctions, such as the N-P, P-M or HN-L junction. For example, a uniqueNot I site was also introduced into the antigenomic HPIV1 cDNA in theP-M junction and this was used to introduce a gene unit expressing theHMPV CAN83 strain G protein.

To generate the HPIV1 based HMPV glycoprotein expression vector, theHMPV CAN83 strain (GenBank Accession No. AY297749 incorporated herein byreference) F glycoprotein ORF was modified for insertion into the Mlu Isite of rHPIV1 described previously (U.S. patent application Ser. No.10/302,547; PCT Publication Number WO 03/043587). The strategy was toexpress the heterologous ORF as an additional, separate mRNA, and henceit was important that it be introduced into the rHPIV1 genome so that itwas preceded by a functional HPIV1 gene start signal and followed by afunctional HPIV1 gene end signal. The cDNA was designed so that theentire inserted gene unit conformed to the “rule of six” and the phasingof the HPIV1 gene start signals were maintained. The Mlu I insertionsite followed the putative gene start signal of the N gene. Hence, forinsertion at this site, the HMPV F ORF needed to be modified byinsertion of an Mlu I site at its upstream end and addition of a HPIV1gene end signal, intergenic region, gene start signal, and a MluI siteat its downstream end (FIG. 14). The inserted sequence was 1656nucleotides in length and thus the length of the modified HPIV1antigenomic cDNA conformed to the rule of six, which holds for othermembers of Genus Respirovirus (Chanock et al., In Fields Virology1:1341-1379, 2001) and also appears to apply to HPIV1.

Recombinant virus (rHPIV1-F₈₃) was readily recovered from transfectedHEp-2 cells using the HPIV1 N, P and L protein expression plasmids andMVA-T7 infection. The virus supernatant was then passaged several timeson LLC-MK2 cells grown at 32° C. vRNA isolated from LLC-MK2 cellsinfected with rHPIV1-F₈₃ was used to generate a RT-PCR product flankingthe supernumerary gene, and sequence analysis confirmed that thesequence of the supernumerary gene present in rHPIV1-F₈₃ was asdesigned. Thus, an additional gene encoding a foreign antigen can bereadily inserted into recombinant HPIV1 using the putative transcriptionsignals and insertion strategy identified in the present example, andthis inserted sequence is stably maintained following prolongedreplication in tissue culture cells.

A similar strategy was used to generate an HPIV1 vector expressing theHMPV G protein. In this example, a unique NotI site was introduced byPCR mutagenesis at nts 3609-3616, between the HPIV1 P and M ORFs, withinthe P gene non-coding region. Then the HMPV CAN83 strain G glycoproteinORF (GenBank Accession No. AF457102) was modified for insertion into theNot I site of rHPIV1. The strategy was to express the heterologous ORFas an additional, separate mRNA, and hence it was important that it beintroduced into the rHPIV1 genome so that it was preceded by afunctional HPIV1 gene start signal and followed by a functional HPIV1gene end signal. The cDNA was designed so that the entire inserted geneunit conformed to the “rule of six” and the phasing of the HPIV1 genestart signals were maintained. Hence, for insertion at this site, theHMPV G ORF needed to be modified by insertion of a Not I site followedby a putative HPIV1 gene end signal, intergenic region, gene startsignal, the HMPV G ORF, and a Not I site at its downstream end. Theinserted sequence was 702 nucleotides in length and thus the length ofthe modified HPIV1 antigenomic cDNA conformed to the rule of six.Recombinant virus (rHPIV1-G₈₃) was readily recovered from transfectedHEp-2 cells using the HPIV1 N, P and L protein expression plasmids andMVA-T7 infection, as described above. The virus supernatant was thenpassaged several times on LLC-MK2 cells grown at 32° C. vRNA isolatedfrom LLC-MK2 cells infected with rHPIV1-G₈₃ was used to generate anRT-PCR product flanking the supernumerary gene, and sequence analysisconfirmed that the sequence of the supernumerary gene present inrHPIV1-G₈₃ was as designed.

Recombinant rHPIV1-F₈₃ and rHPIV1-G₈₃ expressing the HMPV F protein orHMPV G protein from the supernumerary gene were isolated, biologicallycloned, and found to replicate to high titer, ≧7.7 log₁₀ and 9.0 log₁₀TCID₅₀/ml, respectively. Expression of the HMPV and HPIV1 glycoproteinswas confirmed by indirect immunofluorescence of LLC-MK2 cells infectedwith either HPIV1, HMPV, rHPIV1-F₈₃, or rHPIV1-G₈₃. LLC-MK2 cells grownon glass slides were infected with virus, and approximately 72 hourspost-infection the cells were fixed and permeabilized. Mouse monoclonalanti-HPIV1 HN and hamster polyclonal anti-HMPV antibodies were used todetect the HPIV1 HN and HMPV glycoproteins, respectively, in LLC-MK2cells. Fluorescein isothiocyanate (FITC) conjugated anti-mouse oranti-hamster IgG antibody (Jackson Immunochemicals, PA) was used forindirect immunofluorescence of HMPV or HPIV1 glycoproteins. Thus,expression of the HMPV F or G protein was confirmed (Table 12),indicating that supernumerary genes expressing the HMPV F and Gglycoproteins inserted either upstream of the HPIV1 N gene or betweenthe P and M genes, respectively, are well tolerated and efficientlytranslated. Therefore, these recombinant HPIV1 viruses express theprotective antigens of two human pathogens, HMPV and HPIV1.

Using the methods outlined herein, the HPIV1 and HPIV2 vectors describedherein can now be attenuated by the systematic introduction of deletionand/or point mutations to generate live-attenuated viruses that willprotect against two major pediatric respiratory pathogens, for exampleHPIV1 and HMPV. These rHPIV1-F₈₃ and rHPIV1-G₈₃ viruses could be used asvectors to induce a primary antibody response to HMPV, or to boost aresponse induced by prior infection with HMPV or with another PIV vectorsuch as an HPIV3.

TABLE 12 Detection of HMPV or HPIV1 antigens in LLC-MK2 cells infectedwith recombinant rHPIV1 vectors expressing the HMPV glycoproteins byindirect immunofluorescence Level of immunofluorescence detected using aprimary antiserum directed against: Virus HMPV HPIV1 rHPIVI_(LLC4) 0 +++rHPIV1 F₈₃ +++ +++ rHPIV1 G₈₃ +++ +++ HMPV CAN83 +++ 0 uninfected 0 0The virus used to infect LLC-MK2 cells is indicated. The LLC-MK2monolayers were examined using confocal microscopy. The level ofintensity of the immunofluorescence signal obtained from HMPV infectedversus rHPIV1 F₈₃, or rHPIV1 G₈₃ infected cells was equivalent. A “0”indicates no signal detected. A “+++” indicates a strongimmunofluorescence signal.

Recombinant live attenuated HPIV1 and HPIV2 viruses will be very usefulas vectors to express the protective antigens of other human respiratorypathogens, especially those that cause disease in infancy. Since HPIV1and HPIV2 wild type viruses predominantly cause disease in laterinfancy. i.e., in infants older than six months of age, they can beadministered at four to six months of age to prevent HPIV1- andHPIV2-mediated disease. In contrast, RSV, HMPV, and HPIV3 each causedisease in early as well as later infancy, and vaccination against theseviruses will need to be initiated in the first month of life. Thus, thesequential immunization against RSV, HMPV, HPIV3, HPIV1, and HPIV2 willinvolve giving an RSV, HMPV, and HPIV3 immunogenic composition followedseveral months later by a bivalent HPIV1 and HPIV2 immunogeniccomposition. For this reason, HPIV1 and HPIV2 vaccine administration isuniquely positioned to provide an opportunity to boost an immuneresponse to a previously administered RSV, HMPV, or HPIV3 immunogeniccomposition. Since disease caused by RSV and HPIV3 is more severe andmore frequent than that caused by HPIV1 and HPIV2, the ability of aHPIV1 and HPIV2 vaccine to protect against HPIV1 and HPIV2 and to boostan immune response to RSV (or to HMPV or HPIV3) provides added impetusto develop HPIV1 and HPIV2 candidates, i.e., their dual potential willmake them more attractive immunogenic compositions for industry todevelop and for regulatory agencies to approve. The present exampleillustrates the ability of a rHPIV1 virus expressing the RSV Fglycoprotein to boost the immune response to RSV, an immune responseprimed by prior infection with an attenuated RSV.

A live attenuated RSV, RSV_(248/404), was administered to hamsters andone month later the animals were administered HPIV1 wild type virus (acontrol virus), HPIV1-F (HPIV1 expressing the RSV F glycoprotein), or asecond dose of RSV_(248/404) (Table 13). The animals boosted withrHPIV1-F, but not those boosted with RSV_(248/404) or the HPIV1 wildtype control virus, developed a greater than four fold rise in ELISA andneutralizing antibody titer to RSV. Thus, HPIV1-F was able to boost theimmune response to RSV whereas a second dose of RSV_(248/404) was not,presumably because it was not able to infect the RSV immune animals.Thus, the unique ability of rHPIV1-F_(RSV) to boost the response to RSVstems from its ability to replicate in the presence of RSV immunity,whereas this immunity restricted both the replication and immunogenicityof the second dose of the live attenuated RSV_(248/404). The animalsimmunized with rHPIV1-FRSV developed an immune response to HPIV1, asexpected.

A rHPIV2-F vector should be similarly useful as the rHPIV1-F vector atboosting a response to RSV or other such viruses. Furthermore, each ofthese two viruses should be able to vector other protective antigens ofRSV, HMPV and HPIV3 to expand the number of human pathogens that can beprotected against, using a relatively small number of live-attenuatedviruses.

TABLE 13 Immunization of hamsters with rHPIV1 expressing the RSV Fprotein following immunization with RSV248/404 induces a boost serumantibodies against RSV F Serum Serum HAI neut Serum Serum Serum titertiter IgG RSV G IgG RSV F RSV First (log₂) (log₂) ELISA titer ELISAtiter neutralization immunizing Second to to (log₂) (log₂) titersvirus^(a) immunizing HPIV3 HPIV1 on day: on day: (log₂ 60% PNT): 0virus^(b) 28 56 28 56 28 56 28 56 RSV₂₄₈₁₄₀₄ HPIV1-F ≦1 2.6 10.6 10.39.6 12.8 8.3 ± 0.6 10.4 ± 0.5  RSV_(248/404) HPIV1 ≦1 2.8 11.3 11.1 10.810.1 8.9 ± 0.9 9.6 ± 0.9 RSV_(248/404) RSV_(248/404) ≦1 nd 11.8 11.611.3 11.1 8.9 ± 0.7 9.8 ± 0.4 HPIV1 HPIV1-F ≦1 3.8 8.3 9.8 7.1  9.8 — —— HPIV1-F ≦1 3.2 8.1 8.6 7.1  9.6 — — — HPIV1 ≦1 3.7 7.8 8.3 6.6  6.3 —^(a)Hamsters were immunized intranasally with 10⁶ TCID₅₀ of theindicated virus. ^(b)After 28 days, hamsters were immunized intranasallywith 10⁶ TCID₅₀ of the indicated virus. ^(c)Serum was collected 28 daysand 56 days post-first infection. The serum HPIV3 hemagglutinationinhibition titers (HAI), HPIV1 neutralization titers, RSV F and G ELISAand neutralization titers were determined. The mean titer of each group(log₂) is shown.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications may be practicedwithin the scope of the appended claims which are presented by way ofillustration not limitation. In this context, various publications andother references have been cited within the foregoing disclosure foreconomy of description. Each of these references is incorporated hereinby reference in its entirety for all purposes.

1. An infectious, self-replicating human parainfluenza virus type 2(HPIV2) particle comprising a PIV major nucleocapsid (N) protein, a PIVnucleocapsid phosphoprotein (P), a PIV large polymerase (L) protein anda partial or complete polyhexameric HPIV2 genome or antigenome, whereinthe partial or complete HPIV2 genome or antigenome comprises apolynucleotide providing the 3′leader sequence of a HPIV2 andpolynucleotides encoding HPIV2 N, P and L proteins and thepolynucleotide encoding the L protein includes a mutation at the aminoacid aligning with position 20 of SEQ ID NO: 1 to an amino acid otherthan phenylalanine.
 2. The infectious, self-replicating HPIV2 particleof claim 1, in which the mutation at the amino acid aligning withposition 20 of SEQ ID NO: 1 is to alanine or proline.
 3. The infectious,self-replicating HPIV2 particle of claim 1, in which the polynucleotideencoding the L protein further includes a mutation of the amino acidaligning with position 11 of SEQ ID NO: 4 to an amino acid other thanhistidine.
 4. The infectious, self-replicating HPIV2 particle of claim3, in which the mutation of the amino acid aligning with position 11 ofSEQ ID NO: 4 is to leucine.
 5. The infectious, self-replicating HPIV2particle of claim 1, in which the polynucleotide providing the 3′ leadersequence additionally comprises a mutation at of the 3′ leader sequencefrom thymine to cytosine.
 6. The infectious, self-replicating HPIV2particle of claim 2, in which the polynucleotide providing the 3′ leadersequence additionally comprises a mutation at of the 3′ leader sequencefrom thymine to cytosine.
 7. The infectious, self-replicating HPIV2particle of claim 6, in which the polynucleotide encoding the L proteinfurther includes a mutation of the amino acid aligning with position 11of SEQ ID NO: 4 to leucine.
 8. The infectious, self-replicating HPIV2particle of claim 1, in which the partial or complete HPIV2polyhexameric genome or antigenome further comprises a heterologouspolynucleotide encoding one or more antigenic determinants.
 9. Theinfectious, self-replicating HPIV2 particle of claim 7, in which thepartial or complete HPIV2 polyhexameric genome or antigenome furthercomprises a heterologous polynucleotide encoding one or more antigenicdeterminants.
 10. The infectious, self-replicating HPIV2 particle ofclaim 8, in which the heterologous polynucleotide encodes an antigenicdeterminant of a pathogen selected from the group consisting of humanparainfluenza virus type 1, human influenza virus type 3, measles virus,respiratory syncytial virus subgroup A, respiratory syncytial virussubgroup B, mumps virus, human papillomavirus, human immunodeficiencyvirus type 1, human immunodeficiency virus type 2, herpes simplex virus,cytomegalovirus, rabies virus human metapneumovirus, Epstein Barr virus,a filovirus, a bunyavirus, a flavivirus, an alphavirus, and an influenzavirus.